Satoru Harumiya

CD22 serves as a receptor for soluble IgM

CD22 (Siglec‐2) is a B‐cell membrane‐bound lectin that recognizes glycan ligands containing α2,6‐linked sialic acid (α2,6Sia) and negatively regulates signaling through the B‐cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B‐cell activation similar to the Fc receptor for IgG (FcγRIIB).

Nε-(Carboxymethyl)lysine Modification of Elastin Alters Its Biological Properties: Implications for the Accumulation of Abnormal Elastic Fibers in Actinic Elastosis

Accumulation of degenerated elastic fibers in the sun-exposed skin designated as actinic elastosis is a histological hallmark of photodamaged skin. Previous studies have indicated that the elastic fibers of actinic elastosis interact with lysozyme and are modified by N(ɛ)-(carboxymethyl)lysine (CML), one of the major advanced glycation end products (AGEs). We studied here how CML modification of elastin is involved in the pathogenesis of actinic elastosis. The CML-modified insoluble elastin became resistant to neutrophil elastase digestion, which was reversed by treatment with aminoguanidine, a potent inhibitor of AGE formation. In a temperature-dependent aggregation assay, CML-modified elastin rapidly formed self-aggregates, the size of which was larger than unmodified elastin. The elastic fiber sheets prepared from CML-modified α-elastin showed 3D wider diameter, tortuous appearance, and decreased elasticity on tensile tests. The CML-modified α-elastin, but not unmodified α-elastin, was found to bind to lysozyme in vitro, supporting the immunohistochemical findings that the antibodies for lysozyme and CML reacted simultaneously with the elastic fibers of actinic elastosis and UV-irradiated skin. The glycated elastin is likely to cause the accumulation of abnormally aggregated elastic fibers and unusual interaction with lysozyme in actinic elastosis.

Antisense suppression of collagen VI synthesis results in reduced expression of collagen I in normal human osteoblast-like cells.

A transient increase in collagen VI expression precedes the accumulation of collagen I associated with interleukin-4 (IL-4)-induced mineralization in human osteoblast-like cells. Transfection with an antisense oligonucleotide specific for α1(VI) collagen mRNA was shown to attenuate mRNA levels of collagens VI and I. Incubating IL-4 treated cells with anti-collagen VI antiserum decreased expression of α1(I) mRNA. The results suggest that collagen VI may regulate collagen I expression in the early phase of IL-4-induced mineralization.

Tetrameric Interaction of the Ectoenzyme CD38 on the Cell Surface Enables Its Catalytic and Raft-Association Activities

The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.

Inhibition of hepatic metastasis in mice treated with cell-binding domain of human fibronectin and angiogenesis inhibitor TNP-470

AbstractBackground. To prevent tumor metastasis, we administered the cell-binding domain of fibronectin, in combination with the angiogenesis inhibitor TNP-470, to mice with hepatic metastasis. We then assessed the prevention of tumor metastasis resulting from the inhibition of adhesive interactions and the inhibition of angiogenesis. Methods. A hepatic metastasis model was created by injecting 1 × 103 colon 26/TC-1 cells into the anterior mesenteric vein of CDF1 mice. The cell-binding domain obtained from fibronectin included the Arg-Gly-Asp (RGD) sequence. A fibronectin-binding domain (FND)-treated group, an FND plus TNP-470 group, and a control group were established. The animals were killed 4 weeks after the injections of the treatment agents had been completed and the number of metastatic liver nodules was counted. In a simultaneous experiment with the same design, the mice were not killed at 4 weeks, and their survival was observed. Results. The mean number of nodules in the FND plus TNP-470 group was significantly lower than that in the control group (P = 0.019337). The inhibition rate was 51% in the FND group, 60% in the FND 10 μg plus TNP-470 10 mg/kg group, and 64% in the FND 10 μg plus TNP-470 100 mg/kg group compared with the control group. Mice from the FND group that were not killed died after 6–8 weeks, but mice from the FND plus TNP-470 group died after 8–12 weeks. Conclusion. The cell-binding domain of fibronectin may, potentially, be an effective form of antiadhesive therapy that competes with native adhesion molecules and blocks adhesion during the metastatic process. When the cell-binding domain of fibronectin is combined with TNP-470 to inhibit angiogenesis, more effective inhibition of metastatic tumor growth and prolongation of survival can be achieved than after treatment with the cell-binding domain alone.

Galectin‐7 and actin are components of amyloid deposit of localized cutaneous amyloidosis

The precursor protein of localized cutaneous amyloidosis (LCA) is believed to be cytokeratins on the basis of previous immunohistochemical studies. To identify the candidate amyloid protein biochemically, amyloid proteins were extracted with distilled water from lesional skin of LCA associated with Bowens disease. The proteins were resolved on one‐ or two‐dimensional polyacrylamide gel electrophoresis followed by characterization with immunoblot analysis. The proteins with multiple molecular weights of 50–67 kDa and two proteins with 25 and 35 kDa were identified as keratins, serum amyloid P component and apolipoprotein E, respectively. The unknown 14‐kDa (pI = 7.0) and 42‐kDa (pI = 5.4) proteins reacted with the antibody against galectin‐7 and actin, respectively. The protein with the molecular weight of 14 kDa was identified as galectin‐7 by MALDI‐TOF mass spectrometer. Their electrophoretic mobilities were identical with normal counterparts extracted from cultured normal human keratinocytes. Galectin‐7 and actin were detected by immunoblot assay in the water‐soluble fractions prepared from the lesional skins of two patients with primary LCA. Immunohistochemical studies of tumor‐associated (n = 9) and primary (n = 10) LCA revealed various degrees of positive immunoreactivities with the antibodies for galectin‐7 and F‐actin. Galectin‐7 and actin, which contain considerable amount of β‐sheet structure, may be candidate amyloidogenic proteins of primary and secondary LCA.

Involvement of type VI collagen in interleukin-4-induced mineralization by human osteoblast-like cells in vitro.

We recently showed that interleukin-4 (IL-4) enhanced collagen and osteocalcin accumulation and caused mineralization in human periosteal osteoblast-like (SaM-1) cells. At that time, the expression of alpha1(VI) collagen mRNA was induced. In the present study, the possible role of IL-4-induced type VI collagen in the in vitro mineralization in osteoblasts was investigated. Addition of IL-4 in the early stage (for the first 10 days) was essential for the mineralization. The mRNA levels of alpha1(VI) and alpha2(VI) collagen and protein level of type VI collagen were transiently increased by IL-4 treatment up to day 5, whereas the alpha1(I) procollagen mRNA level was greater at day 10 than at day 5. Addition of anti-type VI collagen antibody remarkably reduced the extracellular accumulations of calcium and hydroxyproline induced by IL-4. Furthermore, the transfection of antisense oligonucleotides of alpha1(VI) to SaM-1 cells in the presence of IL-4 partially inhibited IL-4-induced type I collagen accumulation. These results demonstrated that type VI collagen played important roles for IL-4-induced mineralization and hydroxyproline accumulation mostly type I collagen accumulation, in human periosteal osteoblast-like cells.

A system for reconstructing B cell antigen receptor signaling in the mouse myeloma J558L cell line.

B cell antigen receptor (BCR) signaling is positively and negatively regulated by various cell surface receptors such as CD19 and CD45. Functional analysis of these receptors has been performed using gene targeting technology, which is a valid approach to elucidate their functions. However, this type of analysis is restricted when multiple molecules are evaluated simultaneously. From a different perspective, synthetic biology provides a high degree of freedom for analyzing various molecules. Here we developed a system to reconstruct BCR signaling using the J558L myeloma cell line in combination with the protein-based Ca(2+) indicator YC3.60. BCR-reconstituted J558L cells harboring YC3.60 (J558Lμv11 cells) permitted monitoring of Ca(2+) mobilization. Reconstituting CD19 in J558Lμv11 cells resulted in detectable BCR-induced Ca(2+) mobilization but with kinetics different from that of CD45-expressing cells. Furthermore, we evaluated the validity of the J558L system by proteomic analysis of tyrosine-phosphorylated proteins after antigen stimulation. Identification of more than 100 BCR-induced tyrosine-phosphorylated proteins in J558Lμv11 cells revealed a similarity to that observed in B cells, and a novel member, non-receptor protein tyrosine kinase Fer, was found. Thus, this reconstruction system using J558L cells appeared to be valid for comprehensively investigating BCR signaling.