Koichi Honke

Cytokine gene expression in peripheral blood mononuclear cells during graft-versus-host disease after allogeneic bone marrow transplantation

Summary. Cytokine gene expression in peripheral blood mononuclear cells during the development of graft‐versus‐host disease (GVHD) in patients who underwent allogeneic bone marrow transplantation (allo BMT) was analysed using a semiquantitative reverse‐transcriptase polymerase chain reaction (RT‐PCR). The expression of interleukin (IL)‐lβ, IL‐6, and tumour necrosis factor (TNF)‐α mRNA was increased during the development of GVHD and the degree of this increment depended on the severity of the disease. IL‐2 expression was not detected at all and interferon‐γ expression was not much changed during GVHD. In patients with hepatic veno‐occlusive disease (VOD), another transplantation‐related complication, the expression of IL‐1β and TNF‐a mRNA was increased but IL‐6 mRNA expression showed little increase. These findings suggest that IL‐lβ, IL‐6 and TNF‐α produced by peripheral blood mononuclear cells play an important role in the development of GVHD. Furthermore, liver dysfunction due to GVHD or VOD may be distinguishable by this type of cytokine analysis. Analysis of cytokine mRNA expression in peripheral blood mononuclear cells after allogeneic bone marrow transplantation may provide important information concerning the immune response and the cytokine network system in marrow transplant patients.

An assay method for ganglioside synthase using anion-exchange chromatography

A rapid procedure which is based on combined ion-exchange chromatography and solubility was established for determination of the activity of ganglioside synthases and cerebroside sulfotransferase. The procedure consists of selective elution of radiolabeled reaction products (acidic glycolipids) freed from labeled precursors and breakdown products on a DEAE-Sephadex column and of direct radioassay of the products in the eluate. Monosialogangliosides were eluted from the column with 40 mM ammonium acetate (AcONH4) in methanol, cerebroside sulfate with 90 mM AcONH4 in methanol, and disialogangliosides with 40 mM AcONH4 in isopropanol/n-hexane/water (55/20/19, v/v/v). The established procedure is simple, reproducible, and economical. Using rat Golgi membrane as enzyme source the recovery rate of the products was over 95%.

Proteolytic processing of human lysosomal arylsulfatase A

Arylsulfatase A purified from human placenta contained an unreported component with an apparent molecular mass of 7 kDa in addition to the two known components with apparent molecular masses of 58 and 50 kDa. The detailed relationship between the 58 kDa component and the 50 kDa component is as yet unknown. The present study was undertaken to define the structure of the subunits of the sulfatase. The N-terminal sequence of the 50 kDa component was identical to that of the 58 kDa component. Furthermore, the peptide maps of the 50 kDa component, which was separately digested with trypsin and Achromobacter proteinase I, were quite similar to those of the 58 kDa one. Through sequence analysis of the incompatible peaks in the peptide maps, the 50 kDa component was found to lack a sequence from Val-445 to the C-terminus. On the other hand, the N-terminal sequence of the 7 kDa component began with Ala-448, though there was a minor sequence commencing with Thr-449. These observations suggest that the 50 and 7 kDa components were produced by limited proteolysis near the C-terminus of the 58 kDa component. Through analysis using unreducing SDS-PAGE, the 58 and the 7 kDa components were found to be linked by disulphide bonds. Arylsulfatase A purified from human liver was also composed of the same subunits as the placental one. This finding suggests that human arylsulfatase A undergoes similar proteolytic processing regardless of the tissue involved.

Regulation of glycolipid sulfotransferase by tyrosine kinases in human renal cancer cells

Glycolipid sulfotransferase activity in a human renal cancer cell line, SMKT-R3, is enhanced by the action of growth factors such as EGF, TGF-alpha and HGF, whose receptors possess tyrosine kinase domains. We investigated whether tyrosine kinases are involved in the regulation of the sulfotransferase in the cells by using specific tyrosine kinase inhibitors. Genistein and tyrphostin 51 not only cancelled the enhancement of the sulfotransferase by EGF but also reduced the enzyme level to a point much lower than that seen in non-treated cells, whereas they did not affect the sulfotransferase activity in vitro. The activity-reducing effects of genistein were dose- and time-dependent. Genistein also inhibited the cell growth of SMKT-R3 cells. Western blotting using anti-phosphotyrosine monoclonal antibody revealed a tyrosine-phosphorylated protein with an apparent molecular mass of 116 kDa in the non-treated cells. The EGF receptor was tyrosine-phosphorylated by the addition of EGF. The phosphorylations of the 116 kDa protein and EGF receptor were attenuated by co-incubation with genistein. These results indicate that tyrosine kinases including the EGF receptor are involved in the growth of SMKT-R3 cells and in the regulatory mechanisms of glycolipid sulfotransferase in the cells.

Components and proteolytic processing sites of arylsulfatase B from human placenta

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.

INVOLVEMENT OF RAS IN THE EXPRESSION OF GLYCOLIPID SULFOTRANSFERASE IN HUMAN RENAL CANCER CELLS

Glycolipid sulfotransferase activity in a human renal cell carcinoma cell line, SMKT‐R3, is enhanced by epidermal growth factor (EGF); tyrosine kinase inhibitors suppress this enhancement. To investigate the involvement of Ras in the signal transduction pathway from the EGF receptor to the expression of glycolipid sulfotransferase, we introduced v‐H‐ras into SMKT‐R3 cells. In a quiescent state, the percent GTP bound to Ras in v‐H‐ras‐expressing cells increased about 2.5‐fold compared with control cells, suggesting that v‐Ras introduced into the renal cancer cells is in an active form without EGF stimulation. Glycolipid sulfotransferase activity in v‐H‐ras‐expressing cells was higher than in control cells. The sulfotransferase activity was affected neither by EGF nor by genistein, a tyrosine kinase inhibitor, in v‐H‐ras‐expressing cells, whereas it was enhanced by EGF and reduced by genistein in control cells. Our observations suggest that Ras mediates the regulation pathway of glycolipid sulfotransferase activity in SMKT‐R3 cells. Int. J. Cancer 71:620‐623, 1997.

An adult-type metachromatic leukodystrophy caused by substitution of serine for glycine-122 in arylsulfatase A

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase A (ASA). We have identified a new mutation in the ASA gene of a patient with adult-type MLD. In this mutation, the glycine at position 122, a highly conserved residue in the AS gene family, was replaced by serine. In a transient expression study, COS cells transfected with the mutant cDNA carrying 122Gly→Ser did not show an increase of ASA activity and produced little material immunoreactive to an anti-ASA antibody, despite normal mRNA levels.

Pyridoxal 5′-phosphate binds to a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5′-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5′-phosphate followed by reduction with NaBH4 resulted in an irreversible inactivation of the enzyme. When adenosine 3′-phosphate 5′-phosphosulfate was co-incubated with pyridoxal 5′-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5′-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3′-phosphate 5′-phosphosulfate with aKi value of 287 µm. These results suggest that pyridoxal 5′-phosphate modified a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate-recognizing site of the sulfotransferase.

Expression, purification and binding to the receptor of human insulin-like growth factor II

Human insulin-like growth factor II (IGF-II) was expressed as a fused protein with 14 additive amino acids in Escherichia coli with a high yield by an expression system using T7 RNA polymerase. Purification of the expressed protein was simply performed using only differential ultrafiltrations, giving a homogeneous preparation upon polyacrylamide gel electrophoresis and high-performance liquid chromatography. The expressed peptide was reacted with a monoclonal antibody raised against native IGF-II on a blotted membrane. Furthermore, the peptide was bound to IGF-II receptor in solubilized rat fetus membrane, though the affinity was slightly inferior to that of native IGF-II. In addition, fusion IGF-II immobilized on a gel matrix was useful for one-step purification of the IGF-II receptor with a high yield from solubilized rat fetus membranes.

S2.28 Mutations in the arylsulfatase a gene of two patients with metachromatic leukodystrophy

K. Honke 1, T. Kobayashi E, R. Kondo 2, S. Tsuji 2 and A. Makita 1 ~Biochemistry Laboratory, Cancer Institute, Hokkaido University School of Medicine, Sapporo 060, Japan; ZDepartment of Neurology, Brain Research Institute, Niigata University, Niigata 951, Japan. Metachromatic leukodystrophy (MLD) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase A (ASA), which catalyzes the desulfation of galactose-3-sulfate present in sulfated glycolipids. We investigated the ASA gene of two MLD patients: one is juvenile type and the other is adult type. The mutation detected in the ASA gene in the juvenile type MLD was the same as reported by Kondo et al. (1991). On the other hand, the mutation observed in the ASA gene of the adult type MLD was a new one. In that mutation glycine at the position of 122, a residue that is highly conserved in the arylsulfatase gene family, was replaced by serine. In a transient expression study, the COS cells transfected with the mutant cDNA carrying XZZGly~Ser did not show an increase of ASA activity and produced little immunoreactive material with anti-ASA antibody despite its normal mRNA level. These results are in agreement with the reduced immunoreactive material in the patient s liver and probably suggest that the substitution for the highly conserved glycine residue makes the ASA protein unstable.