Mutation study of antithrombin: the roles of disulfide bonds in intracellular accumulation and formation of russell body-like structures

Antithrombin (AT) is a major plasma protease inhibitor with three intramolecular disulfide bonds and a deficiency of it is associated with venous thrombosis. Recently, we prepared CHO cells overexpressing a novel mutant, AT(C95R), with a disulfide bond removed, and revealed that this mutant remained for a long time in the endoplasmic reticulum (ER) without being degraded and also accumulated in newly formed membrane structures that resembled Russell bodies (RB) [Tanaka, Y. et al. (2002) J. Biol. Chem. 277, 51058-51067]. In this study, we replaced each of the individual cysteine residues of AT with an arginine and also two paired cysteine residues with arginines. We stably expressed these mutant ATs in CHO cells, and examined the roles of each cysteine residue or disulfide bond in the accumulation of mutant ATs and the formation of RB-like structures. In pulse-chase experiments, the secretion of mutant ATs with single mutations decreased approximately 1/5-1/50 times compared to that of the wild type AT. All of the mutant ATs were retained in the ER and were also found to accumulate in the RB-like structures. On the other hand, the fates of mutant ATs with double mutations fell into two categories. Secretion of mutant AT(C8R,C128R) decreased only approximately 1/2 times and no RB-like structures appeared. Mutants AT(C21R,C95R) and AT(C247R,C430R) exhibited similar secretion kinetics to the mutant ATs with the single mutations and were found in RB-like structures. On a sucrose gradient, all of the mutant ATs that induced RB-like structures migrated as oligomeric structures, whereas wild type AT and AT(C8R,C128R) migrated as monomers. Further, to clarify the morphological pathway through which RB-like structures are formed, we prepared CHO cells in which the expression of AT(C95R) was controlled by the Tet-On system. During expression of AT(C95R), RB-like structures formed through expansion of the ER. These results suggest that the correct folding with each disulfide bond is essential for the secretion of AT and oligomerization of mutant ATs in the ER is involved in the formation of RB-like structures.     

Deletion of the PDGFR-beta gene affects key fibroblast functions important for wound healing

This study provides new perspectives of the unique aspects of platelet-derived growth factor beta-receptor (PDGFR-beta) signaling and biological responses through the establishment of a mutant mouse strain in which two loxP sequences were inserted into the introns of PDGFR-beta genome sequences. Isolation of skin fibroblasts from the mutant mice and Cre recombinase transfection in vitro induced PDGFR-beta gene deletion (PDGFR-betaDelta/Delta). The resultant depletion of the PDGFR-beta protein significantly attenuated platelet-derived growth factor (PDGF)-BB-induced cell migration, proliferation, and protection from H2O2-induced apoptosis of the cultured PDGFR-betaDelta/Delta dermal fibroblasts. PDGF-AA and fetal bovine serum were mitogenic and anti-apoptotic but were unable to induce the migration in PDGFR-beta Delta/Delta fibroblasts. Concerning the PDGF signaling, PDGF-BB-induced phosphorylation of Akt, ERK1/2, and JNK, but not p38, decreased in PDGFR-betaDelta/Delta fibroblasts, but PDGF-AA-induced signaling was not altered. Overexpression of the phospholipid phosphatases, SHIP2 and/or PTEN, inhibited PDGF-BB-induced phosphorylation of Akt and ERK1/2 in PDGFR-betaDelta/Delta fibroblasts but did not affect that of JNK and p38. These results indicate that disruption of distinct PDGFR-beta signaling pathways in PDGFR-betaDelta/Delta dermal fibroblasts impaired their proliferation and survival, but completely inhibits migratory response, and that PDGF-BB-induced phosphorylation of Akt and ERK1/2 possibly mediated by PDGFR-alpha is regulated, at least in part, by the lipid phosphatases SHIP2 and/or PTEN. Thus, the PDGFR-beta function on dermal fibroblasts appears to be critical in PDGF-BB action for skin wound healing and is clearly distinctive from that of PDGFR-alpha in the ligand-induced biological responses and the underlying properties of cellular signaling.     

Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection

We generated transgenic (TG) mice that constitutively express human CD46 (huCD46) and/or TLR-inducible CD150 (huCD150), which serve as receptors for measles virus (MV). These mice were used to study the spreading and pathogenicity of GFP-expressing or intact laboratory-adapted Edmonston and wild-type Ichinose (IC) strains of MV. Irrespective of the route of administration, neither type of MV was pathogenic to these TG mice. However, in ex vivo, limited replication of IC was observed in the spleen lymphocytes from huCD46/huCD150 TG and huCD150 TG, but not in huCD46 TG and non-TG mice. In huCD150-positive TG mouse cells, CD11c-positive bone marrow-derived myeloid dendritic cells (mDC) participated in MV-mediated type I IFN induction. The level and induction profile of IFN-beta was higher in mDC than the profile of IFN-alpha. Wild-type IC induced markedly high levels of IFN-beta compared with Edmonston in mDC, as opposed to human dendritic cells. We then generated huCD46/huCD150 TG mice with type I IFN receptor (IFNAR1)-/- mice. MV-bearing mDCs spreading to draining lymph nodes were clearly observed in these triple mutant mice in vivo by i.p. MV injection. Infectious lymph nodes were also detected in the double TG mice into which MV-infected CD11c-positive mDCs were i.v. transferred. This finding suggests that in the double TG mouse model mDCs once infected facilitate systemic MV spreading and infection, which depend on mDC MV permissiveness determined by the level of type I IFN generated via IFNAR1. Although these results may not simply reflect human MV infection, the huCD150/huCD46 TG mice may serve as a useful model for the analysis of MV-dependent modulation of mDC response.     

Enzymatic synthesis of chondroitin 4-sulfate with well-defined structure

Synthesis of chondroitin sulfate (ChS) with well-defined structure was achieved for the first time by hyaluronidase-catalyzed polymerization. N-Acetylchondrosine (GlcAbeta(1-->3)GalNAc) oxazoline derivatives sulfated at C4 (1a), C6 (1b), and both C4 and C6 (1c) in the GalNAc unit were synthesized as transition state analogue substrate monomers for hyaluronidase (HAase) catalysis. Compound 1a was effectively polymerized by the enzyme, giving rise to synthetic ChS sulfated perfectly at the C4 position in all N-acetylgalactosamine units (Ch4S, 2a) in good yields. Molecular weights (Mn) of 2a ranged from 4000 to 18,400, which were controlled by varying reaction conditions. Compounds 1b and 1c were not catalyzed by the enzyme, affording the corresponding disaccharides through the oxazoline ring-opening without formation of polysaccharides.     

Preserving genome integrity: the DdrA protein of Deinococcus radiodurans R1

The bacterium Deinococcus radiodurans can withstand extraordinary levels of ionizing radiation, reflecting an equally extraordinary capacity for DNA repair. The hypothetical gene product DR0423 has been implicated in the recovery of this organism from DNA damage, indicating that this protein is a novel component of the D. radiodurans DNA repair system. DR0423 is a homologue of the eukaryotic Rad52 protein. Following exposure to ionizing radiation, DR0423 expression is induced relative to an untreated control, and strains carrying a deletion of the DR0423 gene exhibit increased sensitivity to ionizing radiation. When recovering from ionizing-radiation-induced DNA damage in the absence of nutrients, wild-type D. radiodurans reassembles its genome while the mutant lacking DR0423 function does not. In vitro, the purified DR0423 protein binds to single-stranded DNA with an apparent affinity for 3′ ends, and protects those ends from nuclease degradation. We propose that DR0423 is part of a DNA end-protection system that helps to preserve genome integrity following exposure to ionizing radiation. We designate the DR0423 protein as DNA damage response A protein.     

The structure of starch can be manipulated by changing the expression levels of starch branching enzyme IIb in rice endosperm

When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications.     

Capacitation-like alterations in cooled boar spermatozoa: assessment by the chlortetracycline staining assay and immunodetection of tyrosine-phosphorylated sperm proteins

This study was undertaken in order to characterize alterations occurring in cooled boar spermatozoa by a chlortetracycline (CTC) staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Spermatozoa were collected from 10 mature boars, washed and then resuspended in a Tris-citric acid-glucose (TCG) solution. The sperm suspensions were slowly cooled to 4 degrees C over 5 h and held for 2 days. Aliquots of the sperm suspensions were recovered before and after the cooling treatment and then used for the CTC staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Before the cooling treatment, almost all of the spermatozoa stained with CTC were characterized by uniform fluorescence over the whole head (an F pattern: uncapacitated spermatozoa). After the cooling treatment, however, significant higher percentages of spermatozoa exhibited a B pattern with a dark band of diminished fluorescence in the post acrosomal region and a relatively bright fluorescence in the acrosomal region (the pattern of capacitated spermatozoa). Coincidently, a 32 kDa tyrosine-phosphorylated protein appeared in the spermatozoa. However, these alterations occurring in the cooled spermatozoa were attenuated by the supplementation to the sperm suspensions with seminal plasma (20% (v/v)). Additionally, the same alterations were observed in the spermatozoa incubated in a capacitation-supporting medium (a modified Krebs-Ringer bicarbonate; mKRB) for 5 h. These results suggest that cooling could induce capacitation-like alterations in boar spermatozoa that were associated with the tyrosine phosphorylation of the 32 kDa sperm protein.