A��42-to-A��40- and Angiotensin-converting Activities in Different Domains of Angiotensin-converting Enzyme

Amyloid β-protein 1–42 (Aβ42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Aβ. In contrast, Aβ40 is the predominant secreted form of Aβ and recent studies have suggested that Aβ40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Aβ42 to Aβ40, and its inhibition enhances brain Aβ42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628–8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Aβ42 to Aβ40. Interestingly, Aβ42-to-Aβ40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Aβ42-to-Aβ40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Aβ42-to-Aβ40-converting activity of ACE or increasing neurotoxic Aβ42.     

Murine Coronavirus-Induced Subacute Fatal Peritonitis in C57BL/6 Mice Deficient in Gamma Interferon

Gamma interferon-deficient (IFN-γ−/−) mice with a C57BL/6 background were infected intraperitoneally with mouse hepatitis virus strain JHM (JHMV). In contrast to IFN-γ-+/− and IFN-γ+/+ mice, JHMV persisted in IFN-γ−/− mice and induced death during the subacute phase of the infection. Unexpectedly, infected IFN-γ−/− mice showed severe peritonitis accompanying the accumulation of a viscous fluid in the abdominal and thoracic cavities in the subacute phase. Destructive changes of hepatocytes were not observed. Administration of recombinant IFN-γ protracted the survival time of IFN-γ−/− mice after JHMV infection. These results demonstrate that IFN-γ plays a critical role in viral clearance in JHMV infection. They also show that a resultant persistent JHMV infection induces another form of disease in IFN-γ−/− mice, which bears a resemblance to feline infectious peritonitis in cats.     

Preparation and properties of gelatin-immobilized beta-glucosidase from Pyrococcus furiosus

Hyperthermostable beta-glucosidase from Pyrococcus furiosus was enclosed in gelatin gel by cross-linking with transglutaminase. Gelatin-immobilized beta-glucosidase was considerably more thermostable than the native enzyme. Lyophilized immobilisate was stored at 90 degrees C for 1 month without loss of activity. The immobilized beta-glucosidase catalyzed transglucosylation of 5-phenylpentanol with 10.0 equivalent of cellobiose at pH 5.0 and 70 degrees C for 12 h to afford 5-phenylpentyl beta-D-glucopyranoside in 41% yield. The immobilized enzyme was more effective than the native one in transglucosylation. The gelatin-immobilized Pfu-beta-glucosidase recovered from the first run of the reaction was reusable on successive runs.     

Ubc9 is essential for viability of higher eukaryotic cells

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.     

A unique serine-specific elongation factor Tu found in nematode mitochondria

The translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNAs to ribosomes by recognizing the tRNA acceptor and T stems. However, the unusual truncation observed in some animal mitochondrial tRNAs seems to prevent recognition by a canonical EF-Tu. For instance, nematode mitochondria contain tRNAs lacking a T or D arm. We recently found an atypical EF-Tu (EF-Tu1) specific for nematode mitochondrial tRNAs that lack the T arm. We have now discovered a second factor, EF-Tu2, which binds only to tRNAs that lack a D arm. EF-Tu2 seems unique in its amino acid specificity because it recognizes the aminoacyl moiety of seryl-tRNAs and the tRNA structure itself. Such EF-Tu evolution might explain tRNA structural divergence in animal mitochondria.     

Construction of a Library of Human Glycosyltransferases Immobilized in the Cell Wall of Saccharomyces cerevisiae

Fifty-one human glycosyltransferases were expressed in Saccharomyces cerevisiae as immobilized enzymes and were assayed for enzymatic activities. The stem and catalytic regions of sialyl-, fucosyl-, galactosyl-, N-acetylgalactosaminyl-, and N-acetylglucosaminyltransferases were fused with yeast cell wall Pir proteins, which anchor glycosyltransferases at the yeast cell wall glucan. More than 75% of expressed recombinant glycosyltransferases retained their enzymatic activities in the yeast cell wall fraction and will be used as a human glycosyltransferase library. In increasing the enzymatic activities of immobilized glycosyltransferases, several approaches were found to be effective. Additional expression of yeast protein disulfide isomerase increased the expression levels and activities of polypeptide N-acetylgalactosaminyltransferases and other glycosyltransferases. PIR3 and/or PIR4 was more effective than PIR1 as a cell wall anchor when the Pir-glycosyltransferase fusions were expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Oligosaccharides such as Lewis x, Lewis y, and H antigen were successfully synthesized using this immobilized glycosyltransferase library, indicating that the Pir-fused glycosyltransferases are useful for the production of various human oligosaccharides.     

Functional characterization of the Hansenula polymorpha HOC1, OCH1, and OCR1 genes as members of the yeast OCH1 mannosyltransferase family involved in protein glycosylation

The alpha-1,6-mannosyltransferase encoded by Saccharomyces cerevisiae OCH1 (ScOCH1) is responsible for the outer chain initiation of N-linked oligosaccharides. To identify the genes involved in the first step of outer chain biosynthesis in the methylotrophic yeast Hansenula polymorpha, we undertook the functional analysis of three H. polymorpha genes, HpHOC1, HpOCH1, and HpOCR1, that belong to the OCH1 family containing seven members with significant sequence identities to ScOCH1. The deletions of these H. polymorpha genes individually resulted in several phenotypes suggestive of cell wall defects. Whereas the deletion of HpHOC1 (Hphoc1Delta) did not generate any detectable changes in N-glycosylation, the null mutant strains of HpOCH1 (Hpoch1Delta) and HpOCR1 (Hpocr1Delta) displayed a remarkable reduction in hypermannosylation. Although the apparent phenotypes of Hpocr1Delta were most similar to those of S. cerevisiae och1 mutants, the detailed structural analysis of N-glycans revealed that the major defect of Hpocr1Delta is not in the initiation step but rather in the subsequent step of outer chain elongation by alpha-1,2-mannose addition. Most interestingly, Hpocr1Delta showed a severe defect in the O-linked glycosylation of extracellular chitinase, representing HpOCR1 as a novel member of the OCH1 family implicated in both N- and O-linked glycosylation. In contrast, addition of the first alpha-1,6-mannose residue onto the core oligosaccharide Man8GlcNAc2 was completely blocked in Hpoch1Delta despite the comparable growth of its wild type under normal growth conditions. The complementation of the S. cerevisiae och1 null mutation by the expression of HpOCH1 and the lack of in vitro alpha-1,6-mannosyltransferase activity in Hpoch1Delta provided supportive evidence that HpOCH1 is the functional orthologue of ScOCH1. The engineered Hpoch1Delta strain with the targeted expression of Aspergillus saitoi alpha-1,2-mannosidase in the endoplasmic reticulum was shown to produce human-compatible high mannose-type Man5GlcNAc2 oligosaccharide as a major N-glycan.     

Chimeric analysis of EGFP and DsRed2 transgenic mice demonstrates polyclonal maintenance of pancreatic acini

The pancreatic islet is an assembly of specific endocrine cells. There are many conflicting reports regarding whether the acinus develops from single or multiple progenitor cells. This study investigated the development and maintenance clonality of the pancreatic acinus and duct using a chimeric analysis with EGFP and DsRed2 transgenic mice. Chimeric mice (G-R mice) were obtained by the aggregation method, using 8-cell stage embryos from EGFP and DsRed2 transgenic mice. The islets from the G-R mice were chimeric and mosaic, consisting of either EGFP- or DsRed2-positive populations, as in previous reports. On the other hand, most acini developed from either EGFP or DsRed2 origin, but some were chimeric. Interestingly, these chimeric acini were clearly separated into two-color regions and were not mosaic. Some large intralobular pancreatic ducts consisting of more than 10 cells were found to be chimeric, but no small ducts made up of less than 9 cells were chimeric. Our histological observations suggest that the pancreatic acinus polyclonally and directionally is maintained by multiple progenitor cells. Pancreatic large ducts also seem to develop polyclonally and might result from the assembly of small ducts that develop from a single origin. These findings provide useful information for further understanding pancreatic maintenance.