Akifumi Mizutani

Expression of matrix metalloproteinases during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells.

The mouse calvarial osteoblast MC3T3‐E1 cells released 92 kDa and 68 kDa of gelatinase activities into the conditioned media (CMs) from undifferentiated cells. When differentiation was induced by cultivating cells with ascorbate‐2‐phosphate (AscP), 68‐kDa activity increased significantly in parallel with production of 60‐kDa activity. These enzymes required Ca2+ and Zn2+ ions for their proteolytic activities. The 68‐kDa activity was immunologically identified as latent matrix metalloproteinase 2 (MMP‐2). The 92‐kDa activity was deduced to be latent MMP‐9 based on its molecular mass. The 60‐kDa activity band was found to possess both gelatin and β‐casein hydrolyzing activities, indicating that this activity band might comprise the active form of MMP‐2 and latent MMP‐13. MC3T3‐E1 cells were found to express MMP‐2, MMP‐13, and membrane type (MT)1‐MMP genes by Northern blotting. MMP‐2 was expressed constitutively. MMP‐13 was up‐regulated during the growth with AscP. MT1‐MMP expression also was modulated by AscP; at the early stage of differentiation, its messenger RNA (mRNA) level increased and then decreased gradually to the control level. These changes in the profiles of MMPs observed here could be attributed to the maturation of collagenous extracellular matrix (ECM) induced by AscP.

Topogenesis of two transmembrane type K+ channels, Kir 2.1 and KcsA

Potassium channels, which control the passage of K+ across cell membranes, have two transmembrane segments, M1 and M2, separated by a hydrophobic P region containing a highly conserved signature sequence. Here we analyzed the membrane topogenesis characteristics of the M1, M2, and P regions in two animal and bacterial two-transmembrane segment-type K+ channels, Kir 2.1 and KcsA, using an in vitro translation and translocation system. In contrast to the equivalent transmembrane segment, S5, in the voltage-dependent K+ channel, KAT1, the M1 segment in KcsA, was found to have a strong type II signal-anchor function, which favors the Ncyt/Cexo topology. The N-terminal cytoplasmic region was required for efficient, correctly orientated integration of M1 in Kir 2.1. Analysis of N-terminal modification by in vitro metabolic labeling showed that the N terminus in Kir 2.1 was acetylated. The hydrophobic P region showed no topogenic function, allowing it to form a loop, but not a transmembrane structure in the membrane; this region was transiently exposed in the endoplasmic reticulum lumen during the membrane integration process. M2 was found to possess a stop-transfer function and a type I signal-anchor function, enabling it to span the membrane. The C-terminal cytoplasmic region in KcsA was found to affect the efficiency with which the M2 achieved their final structure. Comparative topogenesis studies of Kir 2.1 and KcsA allowed quantification of the relative contributions of each segment and the cytoplasmic regions to the membrane topology of these two proteins. The membrane topogenesis of the pore-forming structure is discussed using results for Kir 2.1, KcsA, and KAT1.

Defining membrane spanning domains and crucial membrane-localized acidic amino acid residues for K⁺ transport of a Kup/HAK/KT-type Escherichia coli potassium transporter

Potassium (K(+))-uptake transport proteins present in prokaryote and eukaryote cells are categorized into two classes; Trk/Ktr/HKT, K(+) channel, and Kdp belong to the same superfamily, whereas the remaining K(+)-uptake family, Kup/HAK/KT has no homology to the others, and neither its membrane topology nor crucial residues for K(+) uptake have been identified. We examined the topology of Kup from Escherichia coli. Results from the reporter fusion and cysteine labeling assays support a model with 12 membrane-spanning domains. A model for proton-coupled K(+) uptake mediated by Kup has been proposed. However, this study did not show any stimulation of Kup activity at low pH and any evidence of involvement of the three His in Kup-mediated K(+) uptake. Moreover, replacement of all four cysteines of Kup with serine did not abolish K(+) transport activity. To gain insight on crucial residues of Kup-mediated K(+) uptake activity, we focused on acidic residues in the predicted external and transmembrane regions, and identified four residues in the membrane regions required for K(+) uptake activity. This is different from no membrane-localized acidic residues essential for Trk/Ktr/HKTs, K(+) channels and Kdp. Taken together, these results demonstrate that Kup belongs to a distinct type of K(+) transport system.

Genetic modification of a chicken expression system for the galactosylation of therapeutic proteins produced in egg white

As a tool for large scale production of recombinant proteins, chickens have advantages such as high productivity and low breeding costs compared to other animals. We previously reported the production of erythropoietin, the tumor necrosis factor receptor fused to an Fc fragment, and an Fc-fused single-chain Fv antibody in eggs laid by genetically manipulated chickens. In egg white, however, the incomplete addition of terminal sugars such as sialic acid and galactose was found on N-linked glycans of exogenously expressed proteins. This could be a draw back to the use of transgenic chickens since the loss of these terminal sugars may affect the functions and stability of recombinant proteins purified from chicken egg white for pharmaceutical usage. To overcome this problem, we studied galactosyltransferase (GalT) activity in the magnum where the majority of egg-white proteins are secreted. In the magnum, lower β1,4-GalT1 expression and poor galactose-transfer activity were observed. Thus, we supposed that the lack of GalT1 activity may partly cause the incomplete glycosylation of egg-white proteins, and generated genetically manipulated chickens expressing GalT1 by retrovirus-mediated gene transfer. In a Golgi fraction prepared from magnum cells of the genetically manipulated chickens, significant GalT activity was detected. The series of analyses revealed a considerable improvement in the galactosylation of native egg-white proteins as well as an exogenously expressed single-chain Fv antibody fused to an Fc fragment. We conclude that chickens with genetically modified GalT activity in the magnum could be an attractive platform for producing galactosylated therapeutics.

Transcription Factor YY1 Interacts with Retroviral Integrases and Facilitates Integration of Moloney Murine Leukemia Virus cDNA into the Host Chromosomes

ABSTRACT Retroviral integrases associate during the early viral life cycle with preintegration complexes that catalyze the integration of reverse-transcribed viral cDNA into the host chromosomes. Several cellular and viral proteins have been reported to be incorporated in the preintegration complex. This study demonstrates that transcription factor Yin Yang 1 binds to Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. The results of coimmunoprecipitation and in vitro pulldown assays revealed that Yin Yang 1 interacted with the catalytic core and C-terminal domains of Moloney murine leukemia virus and human immunodeficiency virus type 1 integrases, while the transcriptional repression and DNA-binding domains of the Yin Yang 1 molecule interacted with Moloney murine leukemia virus integrase. Immunoprecipitation of the cytoplasmic fraction of virus-infected cells followed by Southern blotting and chromatin immunoprecipitation demonstrated that Yin Yang 1 associated with Moloney murine leukemia virus cDNA in virus-infected cells. Yin Yang 1 enhanced the in vitro integrase activity of Moloney murine leukemia virus, human immunodeficiency virus type 1, and avian sarcoma virus integrases. Furthermore, knockdown of Yin Yang 1 in host cells by small interfering RNA reduced Moloney murine leukemia virus cDNA integration in vivo, although viral cDNA synthesis was increased, suggesting that Yin Yang 1 facilitates integration events in vivo. Taking these results together, Yin Yang 1 appears to be involved in integration events during the early viral life cycle, possibly as an enhancer of integration.

Ascorbate-dependent expression of ubiquitin genes in guinea pigs

In an attempt to characterize a gene(s), of which the expression is ascorbate-dependent, a cDNA fragment encoding ubiquitin was isolated from a subtracted cDNA library constructed from spleen RNAs of ascorbate-deficient or -replete guinea pigs. On Northern blot analysis, three transcripts (1.8 kb ubiX, 1.3 kb ubiY and 0.7 kb ubiZ) were detected. The ubiY encodes four direct repeats of the 76 amino acid ubiquitin sequence with seven additional amino acids, V-Y-A-S-P-I-F, at the C-terminus. The transcript ubiX appears to comprise more than five repeats of the ubiquitin-encoding unit. The ubiZ encodes a ubiquitin monomer fused to an 80 amino acid extension exhibiting 100% amino acid sequence identity to the human homolog, HUMUBA80R. The ubiX gene was expressed animal-dependently. The ubiY mRNA levels decreased under ascorbate-deficient conditions, and increased under ascorbate-replete conditions, whereas ubiZ mRNA remained unaltered at low levels under the feeding conditions used here.

Analyses of chicken sialyltransferases related to N-glycosylation

Proteins exogenously expressed and deposited in the egg whites of transgenic chickens did not contain terminal sialic acid in their N-glycan. Since this sugar is important for the biological stability of therapeutic proteins, we examined chicken sialyltransferases (STs). Based on homologies in DNA sequences, we cloned and expressed several chicken STs, which appeared to be involved in N-glycosylation in mammals, in 293FT cells. Enzymatic activity was detected with ST3Gal3, ST3Gal6 and ST6Gal1 using galactose-β1,4-N-acetylglucosamine (Galβ1,4GlcNAc) as an acceptor. Using Golgi fractions from the cell-free extracts of chicken organs, α2,3- and/or α2,6-ST activities were detected in the liver and kidney, but were absent in the oviduct cells in which egg-white proteins were produced. This result suggested that the lack of ST activities in oviduct cells mainly caused the lack of sialic acid in the N-glycan of proteins exogenously expressed and deposited in egg white.