Masahiro Kajiwara

Ligand-induced structural changes of the CD44 hyaluronan-binding domain revealed by NMR.

CD44, a major cell surface receptor for hyaluronan (HA), contains a functional domain responsible for HA binding at its N terminus (residues 21-178). Accumulating evidence indicates that proteolytic cleavage of CD44 in its extracellular region (residues 21-268) leads to enhanced tumor cell migration and invasion. Hence, understanding the mechanisms underlying the CD44 proteolytic cleavage is important for understanding the mechanism of CD44-mediated tumor progression. Here we present the NMR structure of the HA-binding domain of CD44 in its HA-bound state. The structure is composed of the Link module (residues 32-124) and an extended lobe (residues 21-31 and 125-152). Interestingly, a comparison of its unbound and HA-bound structures revealed that rearrangement of the β-strands in the extended lobe (residues 143-148) and disorder of the structure in the following C-terminal region (residues 153-169) occurred upon HA binding, which is consistent with the results of trypsin proteolysis studies of the CD44 HA-binding domain. The order-to-disorder transition of the C-terminal region by HA binding may be involved in the CD44-mediated cell migration.

Hyaluronan Recognition Mode of CD44 Revealed by Cross-saturation and Chemical Shift Perturbation Experiments

CD44 is the main cell surface receptor for hyaluronic acid (HA) and contains a functional HA-binding domain (HABD) composed of a Link module with N- and C-terminal extensions. The contact residues of human CD44 HABD for HA have been determined by cross-saturation experiments and mapped on the topology of CD44 HABD, which we elucidated by NMR. The contact residues are distributed in both the consensus fold for the Link module superfamily and the additional structural elements consisting of the flanking regions. Interestingly, the contact residues exhibit small changes in chemical shift upon HA binding. In contrast, the residues with large chemical shift changes are localized in the C-terminal extension and the first α-helix and are generally inconsistent with the contact residues. These results suggest that, upon ligand binding, the C-terminal extension and the first α-helix undergo significant conformational changes, which may account for the broad ligand specificity of CD44 HABD.

Mechanism of the ring contraction process in vitamin B12 biosynthesis by the anaerobe Propionibacterium shermanii under aerobic conditions.

The mechanism of the ring contraction process during vitamin B12 biosynthesis by the anaerobe Propionibacterium shermanii was investigated under both aerobic and anaerobic conditions by means of feeding experiments with δ‐amino[1‐13C]levulinic acid (a biosynthetic intermediate of tetrapyrrole) and δ‐amino[1‐13C,1,1,4‐18O3]levulinic acid in combination with 13C‐NMR spectroscopy. We showed that the characteristic mechanism of the ring contraction process (the generation of precorrin‐3x from formation of the γ‐lactone from the ringu2003A acetate group at C1 and hydroxylation at C20 by molecular oxygen catalyzed by CobG, and the migration of ringu2003D by cleavage of the carbon–oxygen bond at C1 of precorrin‐3x) in the aerobe Pseudomonas denitrificans was not seen in P.u2003shermanii under aerobic conditions, and the mechanism of the ring contraction process in P.u2003shermanii was the same irrespective of the presence or absence of oxygen.

An asymmetric synthesis of L-[2-13C]aspartic acid from sodium [2-13C]acetate

l-[2-13C]Aspartic acid was synthesized by using Dellarias oxazinone labelled with 13C at the 3-position, prepared from phenyl [2-13C]bromoacetate and (S)-2-phenylglycinol, as a chiral glycine equivalent. Phenyl [2-13C]bromoacetate was derived from sodium [2-13C]acetate. Alkylation of the [3-13C]oxazinone with ethyl bromoacetate was achieved with high diastereoselectivity. Finally, sequential deprotection and removal of the chiral auxiliary of the alkylated [3-13C]oxazinone afforded l–[2-13C]aspartic acid. Copyright

CARBON SOURCE DEPENDENCE OF THE RATIO OF δ-AMINOLEVULINIC ACID BIOSYNTHESIS VIA THE C5 AND SHEMIN PATHWAYS IN EUGLENA GRACILIS (EUGLENOPHYCEAE)(1).

The ratio of two biosynthetic pathways was estimated, the C5 and Shemin pathways, to δ‐aminolevulinic acid (ALA, a biosynthetic intermediate of tetrapyrrole) from the 13C‐enrichment ratios (13C‐ER) at the carbon atoms of chl a (after conversion to methyl pheophorbide a) biosynthesized by Euglena gracilis G. A. Klebs when l‐[3‐13C]alanine was used as a carbon source. On the basis of these estimations, we confirmed that ALA was efficiently biosynthesized via both the C5 and Shemin pathways in the plastids of E. gracilis, and we determined that the ratio of ALA biosynthesis via the Shemin pathway was increased in the ratio of 14%–67%, compared with that in our previous d‐[1‐13C]glucose feeding experiment ( Iida et al. 2002 ). This carbon source dependence of the contributions of the two biosynthetic pathways might be related to activation of gluconeogenesis by the amino acid substrate. The methoxy carbon of the methoxycarbonyl group at C‐132 of chl a was labeled with the 13C‐carbon of l‐[methyl‐13C]methionine derived from l‐[3‐13C]alanine via [2‐13C]acetyl coenzyme A (CoA), through the atypical tricarboxylic acid (TCA) cycle, gluconeogenesis, and l‐[3‐13C]serine. The phytyl moiety of chl a was also labeled on C‐P2, C‐P31, C‐P4, C‐P6, C‐P71, C‐P8, C‐P10, C‐P111, C‐P12, C‐P14, C‐P151, and C‐P16 from 13C‐isoprene (2‐[1,2‐methyl,3‐13C3]methyl‐1,3‐butadiene) generated from l‐[3‐13C]alanine via [2‐13C]acetyl CoA.

13C‐phenylalanine breath test correlates with liver fibrosis in postoperative biliary atresia

Background: Values derived from the 13C‐phenylalanine breath test (PBT) may serve as an index for liver fibrosis and clinically predictive readings for liver diseases in adults. In the present study the PBT was conducted in postoperative biliary atresia (BA) children to evaluate phenylalanine metabolism in the liver, and the results based on biochemical data, especially the index on liver fibrosis, were compared with PBT findings.

Metabolic pathways leading from amino acids to vitamin B12 in Propionibacterium shermanii, and the sources of the seven methyl carbons

The metabolic pathways leading from l‐[2‐13C]aspartic acid, [2‐13C]glycine and l‐[methyl‐13C]methionine to vitaminu2003B12 were investigated, focusing on the biosynthetic pathways leading to the aminopropanol moiety of vitaminu2003B12 and on the role of the Shemin pathway leading to δ‐aminolevulinic acid (a biosynthetic intermediate of tetrapyrrole), by means of feeding experiments with Propionibacterium shermanii in combination with 13C‐NMR spectroscopy. The 13C‐methylene carbons of l‐[2‐13C]aspartic acid, which is transformed to [2‐13C]glycine via l‐[2‐13C]threonine, and [2‐13C]glycine added to the culture medium served mainly to enrich the seven methyl carbons of the corrin ring through C‐methylation by S‐adenosyl‐l‐[methyl‐13C]methionine derived from catabolically generated l‐[methyl‐13C]methionine in the presence of tetrahydrofolic acid. The results indicate that the catabolism of these amino acids predominates over pathways leading to (2R)‐1‐amino‐2‐propanol or δ‐aminolevulinic acid in P.u2003shermanii. Feeding of l‐[methyl‐13C]methionine efficiently enriched all seven methyl carbons. In the cases of [2‐13C]glycine and l‐[methyl‐13C]methionine, the 13C‐enrichment ratio of the methyl carbon at C‐25 (the site of the first C‐methylation) was less than those of the other six methyl carbons, probably due to the influence of endogenous d‐glucose in P.u2003shermanii. The almost identical 13C‐enrichment ratios of the other six methyl carbons indicated that these C‐methylations during vitaminu2003B12 biosynthesis were completed before the amino acids were completely consumed. However, in the case of l‐[2‐13C]aspartic acid, the 13C‐enrichment ratios of five methyl carbons were low and similar, whereas the last two sites of C‐methylation (C‐53 and C‐35) were not labeled, presumably because of complete consumption of the smaller amount of added label. The ratios of 13C‐incorporation into the seven methyl carbons are influenced by the conditions of amino acid feeding experiments in a manner that is dependent upon the order of C‐methylation in the corrin ring of vitaminu2003B12.

Effect of Concomitant18O in13C-Urea on the Urea breath test

Helicobacter pylori infection in stomach has been extensively assessed by the 13C-urea breath test (UBT) with infra-red or mass spectrometer. However, analyses of commercially available 13C-urea for UBT showed about 10 atom% of 18O abundance. It is therefore very important to investigate the 18O-isotope effect on UBT. Using a mass spectrometer, breath samples of UBT were analyzed to investigate the change of mass spectra by the isotopic exchange reaction between 18O of carbon dioxide and 16O of water. As the result, the 18O content of 13C, 18O-carbon dioxide exhaled in UBT reached its natural abundance level immediately after the administration of 13C, 18O-urea. It was concluded that the 18O content of 13C, 18O-urea has no effect on UBT using either infra-red or mass spectroscopy. Copyright

A simple convenient synthesis of l‐[4‐13C]glutamine

L-[4-(13)C]Glutamine was synthesized from sodium [2-(13)C]acetate in 12 steps and 18% overall yield. A Wittig reaction of (R)-benzyl 4-formyl-2,2-dimethyloxazolidine-3-carboxylate and ethyl 2-(triphenylphosphoranylidene)[2-(13)C]acetate prepared from D-serine and sodium [2-(13)C]acetate, respectively, gave (4S)-4-(2-ethoxycarbonyl[2-(13)C]vinyl)-2,2-dimethyloxazolidine-3-carboxylic acid α,β-isopropylidene group, oxidation of the resulting hydroxyl group to a carboxyl group and transamidation of the ester moiety gave L-N-Cbz-[4-(13)C]glutamine (Cbzu2009=u2009benzyloxycarbonyl). Finally, removal of the Cbz group gave L-[4-(13)C]glutamine. L-[4-(13)C]Glutamine can be prepared in fewer steps and higher yield by this method compared with previously reported methods.