Mariko Iwasaki

Isolation of a novel glycoprotein from the eggs of rainbow trout: Occurrence of disialosyl groups on all carbohydrate chains

Abstract The occurrence of disialosyl (α-N-glycolylneuraminyl-(2→8)-N-glycolylneuraminyl) groups has been shown in the carbohydrate chains of a glycoprotein newly isolated from the eggs of rainbow trout. Almost all sialic acid residues form disialosyl sequences at the terminal position of the oligosaccharide units consisting of galactose and N-acetylgalactosamine through which the units are linked to threonine residues of the peptide moiety. The sialic acid content of the glycoprotein was as high as 50%. Such a glycoprotein has not been recognized as a constituent of fish eggs. Its unique sugar and amino acid composition is different from that of any class of the glycoproteins so far reported.

Structures of carbohydrate units isolated from trout egg polysialoglycoproteins: short-cored units with oligosialosyl groups.

Abstract The eggs of rainbow trout contain a novel type of glycoproteins in which sialic acid (NeuGc) accounts for about 60% of the weight. Of more than 20 oligosaccharide fractions containing various numbers of NeuGc residues obtained by alkaline borohydride treatment of these polysialoglycoproteins followed by an anion-exchange chromatography, structures of a series of oligosaccharides, Galβ1–3[(NeuGcα2–8) n NeuGcα2–6]GalNAcol ( n = 0–3), have been determined. 1 H-NMR signals for H-3 protons of NeuGc residues in oligosialosyl groups were assigned using model NeuAc oligomers prepared by limited acid hydrolysis of colominic acid.

Evaluation of bidirectional transfer of plasma DNA through placenta

AbstractTo clarify the origin of cell-free fetal DNA in maternal plasma, we analyzed bidirectional transfer of plasma DNA between fetus and mother. We analyzed maternal and fetal plasma DNA obtained from 15 pregnant women at the time of Cesarean section. The subjects were five patients with preeclampsia and 10 gestational-age-matched normal controls. DNA was extracted from 1.5-ml plasma samples and the cellular fraction of maternal and umbilical blood. Seven polymorphic marker genes were analyzed. The relative concentration of fetal DNA in maternal plasma and maternal DNA in cord blood were evaluated. The relative concentration of maternal DNA in fetal circulation (median, 0.9%; range, 0.2–8.4%) was significantly lower than that of fetal DNA in maternal blood (14.3%, 2.3–64%), with P=0.007. The relative concentration of maternal DNA in fetal blood was not affected by preeclampsia. These findings indicate that cell-free DNA is unequally transferred through the placenta. The structural characteristics of the placenta suggest that the majority of cell-free fetal DNA in maternal plasma is derived from villous trophoblasts.

Relationship between Severity of Hyperemesis Gravidarum and Fetal DNA Concentration in Maternal Plasma

Cell-free fetal DNA is present in maternal plasma (1), where its concentration increases during some abnormal conditions that can occur during pregnancy, including preterm delivery (2), preeclampsia (3)(4)(5), invasive placenta (6), and fetal trisomy 21 (7)(8). The origin of fetal DNA is not clear, but a body of evidence in the literature suggests that it comes mainly from the destruction of villous trophoblasts that border the intervillous space filled with maternal blood (5)(9). Although the pathogenesis of hyperemesis gravidarum (HG) is obscure, activation of natural killer and cytotoxic T cells is higher in the blood and uterine decidua of women with HG with respect to healthy pregnant women (10). Thus, if HG and DNA concentrations were correlated, one possible common pathway that could explain these conditions might be an overactivated maternal immune system that destroys trophoblasts, causing both HG and higher concentrations of cell-free fetal DNA. The aim of this study was to investigate a possible correlation between HG and fetal DNA concentrations. We also evaluated the relationship between the cell-free fetal DNA concentration and the severity of HG. A total of 202 pregnant women (gestational age, 6–16 weeks) bearing a single male fetus, who presented at Showa University Hospital between July 2000 and July 2002 were enrolled in this study. Forty-five consecutive pregnant women with HG were classified into three groups based on the severity of the condition and matched for gestational age with 157 controls. All samples were analyzed blindly without knowledge of case-control status. The three HG groups were generated according to the following criteria: mild HG (nausea and vomiting but no need for admission), moderate HG (admission for HG with dehydration that needed infusion therapy but lacking all of the criteria that define severe HG), and severe HG …

Structures of the Carbohydrate Units of Polysialoglycoproteins Isolated from the Eggs of Four Species of Salmonid Fishes

Fish egg polysialoglycoprotein (PSGP) is a novel type of 200 kDa-glycoprotein containing more than 50% sialic acid by weight and about 90O-glycosidically-linked sialoglycan units per molecule. Of about 100 different molecular species assumed to be present in a sialoglycan mixture obtained by alkaline borohydride treatment ofSalvelinus leucomaenis pluvius PSGP, 23 mono- to tetrasialylglycans were isolated by anion-exchange chromatography and preparative column chromatography on porous silica, and their structures were determined. Core asialo-oligosaccharides were obtained from PSGP of four species of salmonid fishes by exhaustive enzymatic desialylation of sialoglycan mixtures and the structures of purified compounds were determined. Two complete types of asialopentasaccharide core structures, Fucα1-3GalNAcβ1-3Galβ1-4 Galβ1-3GalNAcOL, and GalNAcβ1-4GalNAcβ1-3Galβ1-4Galβ1-3GalNAcOL, and all of the possible biosynthetic precursors of these pentasaccharide cores were found in every PSGP examined. All types of oligosaccharide chains, both complete and incomplete, were found to occur in highly sialylated forms in PSGP.

Characterization of a new type of glycoprotein saccharides containing polysialosyl sequence

Summary The occurrence of polysialosyl sequence has been shown for the first time in the carbohydrate units of glycoprotein isolated from the eggs of rainbow trout. The saccharide chains accounting for 80 % of the weight of the glycoprotein were O-glycosidically linked to the peptide moiety. The reduced saccharides obtained by alkaline borohydride treatment of the glycoprotein contain various amounts of N-glycolylneuraminic acid and were fractionated by a column of DEAE-Sephadex. Analyses of the saccharides containing large amounts of N-glycolylneuraminic acid show that 2 → 8 linked homopolymers of more than 15 N-glycolylneuraminic acid residues were attached to the core composed of 2 galactose, 1 N-acetylgalactosamine, and 1 N-acetylgalactosaminitol residues.

Measurement of mRNA of trophoblast-specific genes in cellular and plasma components of maternal blood

Background: Placental mRNA in maternal plasma is suitable for quantitative analysis regardless of fetal gender and genetic polymorphism status. Methods: We obtained 155 blood samples from pregnant women to compare human placental lactogen (hPL) and β-subunit of human chorionic gonadotropin (βhCG) mRNA and protein levels between the cellular and plasma components of maternal blood. To assess clearance of hPL mRNA expression, we obtained blood samples from nine women immediately before and after delivery by caesarean section. mRNA was extracted from the cellular and plasma components of all samples, and hPL and βhCG mRNA expression was analysed by reverse transcription-PCR assay. Results: The concentration of βhCG mRNA in the cellular component positively correlated with the plasma concentration of βhCG protein and βhCG mRNA (p = 0.001 for both). The concentration of hPL protein in the plasma correlated with the hPL mRNA concentration of the cellular component (p<0.05). For both hPL and βhCG, the mRNA concentration of the cellular component was greater than that of the plasma component (22.9-fold higher for hPL and 4.3-fold higher for βhCG). The half life of hPL mRNA clearance was significantly longer for the cellular fraction (mean half life = 203.8 min, range 150–3465 min) than for the plasma fraction (mean half life = 32.2 min, range 15–385 min) (p = 0.008). Conclusion: The present findings indicate that the concentration of hPL and βhCG mRNA is significantly higher in the cellular component of maternal blood samples than in the plasma component. Cellular mRNA in maternal blood is useful for non-invasive evaluation of placental function.

Diagnosis of fetal anomalies by three‐dimensional imaging using helical computed tomography

We present the first report on the use of helical computed tomography (CT), a new, non‐invasive diagnostic technique that produces three‐dimensional (3‐D) images, in prenatal diagnosis. This technique was used to construct 3‐D images in the prenatal diagnosis of two anomalous fetuses. The 3‐D images provided clear information about the anomalies: trisomy 18 in one case and cystic hygroma in the other. In one case, rapid intervention after a planned Caesarean section prevented respiratory distress. Surgery to correct the anomaly was performed 2 days postnatally; the infant recovered uneventfully.

Novel carbohydrate structures in trout egg glycoprotein: Occurrence of a neuraminidase-resistant N-glycolylneuraminosyl-(2→3)-N-acetylgalactosamine linkage

Summary Oligosaccharides with a new type of core structure, GalNAcβ1→4 Galβl→4[NGNA2→3]GalNAcβ1→3Ga1β1→3Ga1NAco1, have been isolated from trout egg glycoprotein. The occurrence of N-glycolylneuraminic acid (NGNA) linked 2→3 to the internal Ga1NAc residue is not known in any other glycoproteins and glycolipids. This NGNA residue was found to show some anomalous nature such as resistance to neuraminidases and unusual chemical shift values of H-3 protons in NMR spectra. A new feature is also found in the asialooligosaccharide structure, Ga1NAcβl→4Galβ1→4Ga1NAc, linked β1→3 to the linkage region disaccharide, Ga1β1→3Ga1NAc.

S1.11 Identification and developmentally regulated expression of acid and alkaline peptide-N4 (N-acetyl-β-glucosaminyl)asparagine amidases (PNGase) inOryzins latipes embryos: The first demonstration of the occurrence of an enzyme responsible for de-N-glycosylation in animal origin

S. Inoue, M. Iwasaki, A. Seko 1, K. Kitajima I and Y. Inoue 1 School of Pharmaceutical Sciences, Showa University, Hatanodai1, Tokyo 142. 1 Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo-7, Tokyo 113, Japan. No report for PNGase from animal source had been found until we identified its presence in the early embryos of Oryzias latipes (1), though PNGase activity has been detected in the extracts of a variety of plant seeds and bacteria. Our previous findings of the accumulation of free N-glycan chains that retain di-N-acetylchitobiosyl structure at the reducing termini at the late stage of oogenesis (2) and blastulation stage of embryogenesis (3) have strongly supported the supposition that PNGase activity is expressed also in animal cells and its expression may be significant for the metabolism and function of certain glycoproteins. Two types of glycoproteins, phosvitin and hyosophorin, have been identified as the physiological substrates of the PNGases. Our current interest is to find if PNGase activity appears stage-specifically during oogenesis and /o r embryogenesis, and to clarify the significance of de-N-glycosylation of phosvitin and hyosophorin in the fish embryos. The carbohydrate units on glycoproteins may serve as recognition sites, regulate conformation of the molecule, and control proteolytic degradation. De-N-glycosylation of a glycopeptide or glycoprotein, which converts the carbohydrate-linked Asn residue to the Asp thereby introducing negative charge and alterating the peptide or protein conformation, may be a possible means to produce a functional conformation. Our preliminary results showed that during O. latipes embryogenesis the PNGase activity appeared to rise rather progressively to a maximum at the late blastula stage, followed a decay. We detected two different PNGase activities in O. latipes early embryos, one is acid PNGase and the other alkaline PNGase. (1) A. Seko, K. Kitajima, Y. Inoue, and S. Inoue (1991) J. Biol. Chem. 266, 22110-22114. (2) M. Iwasaki, A. Seko, K. Kitajima, Y. Inoue, and S. Inoue (1992) J. Biol. Chem. 267, 24287-24296. (3) A. Seko, K. Kitajima, S. Inoue, and Y. Inoue (1991) Biochem. Biophys. Res. Commun. 180, 11651171.