Motonobu Yoshida

A neutral ceramidase homologue from Dictyostelium discoideum exhibits an acidic pH optimum

The nucleotide sequence reported for the Dictyostelium discoideum ceramidase is available on the DNA Data Bank of Japan (DDBJ). Ceramidases (CDases) are currently classified into three categories (acid, neutral and alkaline) based on their optimal pHs and primary structures. Here, we report the first exception to this rule. We cloned the CDase cDNA, consisting of 2142 nucleotides encoding 714 amino-acid residues, from the slime mould, Dictyostelium discoideum. The putative amino-acid sequence indicates 32-42% identity with various neutral CDases, but does not show any similarity to the acid and alkaline CDases, indicating the enzyme should be classified as a neutral CDase. However, overexpression of the cDNA in D. discoideum resulted in increased CDase activity at an acidic, but not a neutral pH range. Knockout of the gene in slime mould eliminated CDase activity at acidic pH. The recombinant enzyme expressed in the slime mould was purified and then characterized. Consequently, the purified CDase was found to exhibit the maximal activity at approx. pH 3.0. The singular pH dependency of slime mould CDase is not derived from the specific post-translational modification in the slime mould, because the enzyme showed an acidic pH optimum even when expressed in Chinese hamster ovary cells, whereas rat neutral-CDase exhibited a neutral pH optimum when expressed in slime mould.

Electrophoretic karyotype of Flammulina velutipes and its variation among monokaryotic progenies

Abstract The karyotype of Flammulina velutipes (Curt. : Fr.) Sing. was investigated using contour-clamped homogeneous electric fields (CHEF) gel electrophoresis. A parental dikaryotic stock, JA, was resolved into at least eight chromosomal DNA bands ranging from 1.4- to 4.9-megabase (Mb) pairs. Overall, little size variation was found among monokaryotic strains with a few major exceptions. Among 13 monokaryotic progenies examined, 11 strains were resolved into at least eight chromosomal DNA bands in a manner similar to the parent dikaryon, whereas the other 2 were resolved into at least seven chromosomes lacking the 2.1-Mb chromosome possessed in the former. A slightly larger size variation was found in a chromosome carrying ribosomal DNA. An estimated haploid genome size of this stock was 24.0 Mb or more.

Characterization of A Glycosylphosphatidylinositol- Anchor in a Cell Adhesion Molecule, Csa, from Dictyostelium Discoideum 1

Abstract The cell adhesion molecule, csA, treated with lysyl endopeptidase led to a peptide fragment with a molecular weight of 31 kDa (31-kDa csA). It was shown after the treatment of the 31-kDa csA with phosphatidylinositol-specific phospholipase C and following extraction with chloroform using TLC that the 31-kDa csA contained a component of glycosylphosphatidylinositol (GPI), and it was also confirmed that the cell adhesion molecule, csA, consisted of ceramide instead of diacylglycerol. The results from GC-MS suggested that hydrocarbons of C16- C33 interacted with the GPI-anchor region. 1. Presented at the XVIII th Intenrarional Carbohydrate Symposium, Milan, Italy, July 21-26, 1996.

Carbohydrate structures of the cell adhesion molecule, contact site A, from Dictyostelium discoideum

We determined the carbohydrate structures of contact site A from Dictyostelium discoideum. The carbohydrate moieties of contact site A were released by hydrazinolysis. Fractionation of the deacidified oligosaccharide mixture by Bio‐Gel P‐4 column chromatography revealed that it was composed of four major oligosaccharides. Their respective structures were determined by sequential exoglycosidase digestion. It is known that contact site A consists of two kinds of carbohydrates, I and II. Taking together the previous and the present results, it was deduced that carbohydrate I comprises N‐linked oligosaccharides and carbohydrate II O‐linked ones. Furthermore, the relative molar contents of GalNAc and GlcNAc in reducing terminal suggested that contact site A contains 67% of N‐linked and 33% of O‐linked oligosaccharides.

Function of the carbohydrates in contact site A glycoprotein of Dictyostelium discoideum affected by tunicamycin.

1. The relationship between glycosylation of contact site A (csA) of 80 kDa with two types of N-linked carbohydrates, I and II, and EDTA-resistant cell contact of Dictyostelium was investigated by tunicamycin treatment. 2. Carbohydrate I glycosylation, involved in a shift of csA from 66 to 80 kDa, was more sensitive to tunicamycin than carbohydrate II glycosylation in its shift from 53 to 66 kDa. 3. The appearance of csA of 80 kDa corresponded to that of EDTA-resistant cell contact. Carbohydrate I may be essential for EDTA-resistant cell contact. 4. In starved cells treated with tunicamycin, only 4-8% of moieties labeled with wheat germ agglutinin in carbohydrate II were modified.

Enzymatic Staining for Detection of Phenol-Oxidizing Isozymes Involved in Lignin- Degradation by Lentinula edodes on Native-PAGE

Lignocellulose is the most abundant organic compound in the terrestrial environment. Nonetheless, with the exception of basidiomycetous fungi, most organisms are either unable to degrade lignocellulose, or if they can, they do so with difficulty (Kirk & Fenn, 1982). Wood-decomposing basidiomycetes can be grouped into two categories: white-rot and brown-rot fungi. White-rot fungi have cellulases and lignin-degrading enzymes that decompose most cell wall components, whereas brown-rot fungi have enzymatic systems that selectively degrade cellulose and hemicelluloses, leaving brown shrunken lumps of tissue composed mainly of a loose lignin matrix (Enoki et al., 1988; Highley et al., 1985; Highley & Murmanis, 1987; Kirk & Highley, 1973). The name ‘white-rot’ is derived from the bleaching effect that this fungus has when degrading wood; the lignin-degrading enzymes that they secrete have the effect of promoting lignin loss and exposing the white cellulose fibrils. White-rot fungi are known to produce polyphenol oxidases (phenoloxidases), which, when the fungi are plated on agar media containing gallic or tannic acids, change the color of the agar to a dark reddish-brown in what is referred to as Bavendamm’s polyphenol oxidase test or Bavendamm reaction (Bavendamm, 1928, as cited in JØrgensen & Vejlby, 1953). Based on this reaction, phenoloxidases are considered to be one of putative lignindegrading enzymes (Higuchi 1990). Laccase (Lcc, EC 1.10.3.2), catechol oxidase (EC 1.10.3.1) and tyrosinase (monophenol monooxygenase, EC 1.14.18.1) are phenoloxidases with considerable overlap in their substrate affinities (Burke & Cairney, 2002). Lcc catalyze the reduction of O2 to H2O using a range of phenolics, aromatic amines, and other electron-rich substances as hydrogen donors (Thurston, 1994). Similar phenol-oxidizing activities are also observed in peroxidases (EC 1.11.1.x), which use H2O2 as an electron donor. Lignin peroxidase (ligninase, LiP, EC 1.11.1.14) was first discovered in Phanerochaete chrysosporium in which the H2O2-dependent C┙-C┚ cleavage of non-phenolic lignin model compounds was first described (Tien & Kirk, 1983, 1984). Manganese peroxidase (MnP, EC 1.11.1.13) also strongly degrades lignin model compounds and the reaction is mediated by H2O2 and Mn2+ (Glenn et al., 1983; Glenn & Gold, 1985; Kuwahara et al., 1984). Whereas lignin can effectively be oxidized by LiP directly, as reviewed previously (Cullen & Kersten, 2004;

Dictyostelium Genes Dysregulated in an O-Glycosylation Mutant Identified by mRNA Differential Display

Seven differentially-expressed cDNA clones were isolated by using an mRNA differential display between a Dictyostelium wild-type AX2 and a mutant HG794 defective in O-glycosylation. Transcript levels for the seven clones were reduced or not detectable in the mutant HG794. Homology search showed that the four cDNA clones, DD-3 and DD-7~9 are novel and that three cDNA clones, DD-4 and DD-5, -6 encode an actin-bundling protein and phosphodiesterase inhibitors, respectively. Full-length cDNAs for DD-3 and -8 were isolated and labeled DD3-3 and DD8-14, respectively. DD3-3 consists of 2,166 bp and DD8-14 of 2,084 bp. DD3-3 was preliminarily reported in a previous paper [1]. SSL850 was named a clone by the “Dictyostelium cDNA Project in Japan”, containing a fulllength cDNA for DD-7 and was labeled DD7-1 of 902 bp. It has 60% homology with discoidin Ia. DD8-14 most likely has no direct role in glycosylation, while DD3-3 and DD7-1 very likely are involved in some aspect of recognition of glycosylation.