Yasuhiko Masuho

Molecular cloning and characterization of a novel type of histamine receptor preferentially expressed in leukocytes.

Recently cDNA encoding the histamine H3 receptor was isolated after 15 years of considerable research. However, several studies have proposed heterogeneity of the H3 receptor. We report here the molecular cloning and characterization of a novel type of histamine receptor. A novel orphan G-protein-coupled receptor named GPRv53 was obtained through a search of the human genomic DNA data base and analyzed by rapid amplification of cDNA ends (RACE). GPRv53 possessed the features of biologic amine receptors and had the highest homology with H3 receptor among known G-protein-coupled receptors. Mammalian cells expressing GPRv53 were demonstrated to bind and respond to histamine in a concentration-dependent manner. In functional assays, not only an H3 receptor agonist,R-(α)-methylhistamine, but also a H3 receptor antagonist, clobenpropit, and a neuroleptic, clozapine, activated GPRv53-expressing cells. Tissue distribution analysis revealed that expression of GPRv53 is localized in the peripheral blood leukocytes, spleen, thymus, and colon, which was totally different from the H3 receptor, whose expression was restricted to the brain. The discovery of the GPRv53 receptor will open a new phase of research on the physiological role of histamine.

Membrane-type 6 matrix metalloproteinase (MT6-MMP, MMP-25) is the second glycosyl-phosphatidyl inositol (GPI)-anchored MMP.

A recently identified membrane‐type 6 matrix metalloproteinase (MT6‐MMP) has a hydrophobic stretch of 24 amino acids at the C‐terminus. This hydrophobicity pattern is similar to glycosyl‐phosphatidyl inositol (GPI)‐anchored MMP, MT4‐MMP, and other GPI‐anchored proteins. Thus, we tested the possibility that MT6‐MMP was also a GPI‐anchored proteinase. Our results showed that MT6‐MMP as well as MT4‐MMP were labeled with [3H]ethanolamine indicating the presence of a GPI unit with incorporated label. In addition, phosphatidyl inositol‐specific phospholipase C treatment released MT6‐MMP from the surface of transfected cells. These results strongly indicate that MT6‐MMP is a GPI‐anchored protein. Since two members of MT‐MMPs are now assigned as GPI‐anchored proteinase, MT‐MMPs can be subgrouped into GPI type and transmembrane type.

Interaction of the Unc-51-like kinase and microtubule-associated protein light chain 3 related proteins in the brain: possible role of vesicular transport in axonal elongation.

We identified two mammalian ULK1 (Unc-51-like kinase involved in neurite extension) binding proteins by yeast two-hybrid screening. Both proteins showed high structural similarity to microtubule-associated protein (MAP) light chain 3 (LC3). One is identical to the Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), an essential factor for intra-Golgi transport [39]. The other is identical to the gamma 2-subunit of GABA-A receptor associated protein (GABARAP) which has a possible role in receptor transport [46]. Using the yeast two-hybrid system and the in vitro GST pull-down assay, we found that the N-terminal proline/serine rich (PS) domain of ULK1 (amino acid 287-416) is required for ULK1-GATE-16 and ULK1-GABARAP protein interactions. However, the kinase activity of ULK1 affected neither ULK1-GATE-16 nor ULK1-GABARAP interaction. Immunohistochemical analysis using ULK1 and GABARAP antibodies showed that the ULK1 and the GABARAP proteins co-localized to many kind of neurons such as pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, and Purkinje cells of the cerebellum. In HeLa cells, endogenous ULK1 and tagged GABARAP showed punctate structures in the cytosol, and were colocalized. These results suggest that the interaction of ULK1 and GABARAP is important to vesicle transport and axonal elongation in mammalian neurons.

The Fibronectin Extra Domain A Activates Matrix Metalloproteinase Gene Expression by an Interleukin-1-dependent Mechanism

The extra domain-A (EDA), present in fibronectin (FN) molecules arising from alternatively spliced transcripts, appears only during specific biological and pathogenic processes. However, its function is poorly understood. To define the physiologic role of this domain in joint connective tissue, the biological effects on rabbit cartilage explants, chondrocytes, and synovial cells were studied. A recombinant EDA protein (rEDA) increased proteoglycan release (3.6-fold) in cartilage explant cultures and markedly induced production of matrix metalloproteinase (MMP)-1 in chondrocytes. In addition, rEDA induced MMP-1, MMP-3, and MMP-9 in synovial cells. These effects were elicited only by rEDA, while its neighboring type III repeats, III11 or III12, scarcely had any such effects. Interestingly, reorganization of F-actin stress fibers accompanied MMP-1 expression in synovial cells treated with rEDA, suggesting alteration of cellular phenotype. Subsequent Northern blotting revealed expression of pro-inflammatory cytokines, including interleukin (IL)-1α and IL-1β, was induced by rEDA prior to MMP-1 expression. Delayed MMP-1 expression suggests that rEDA-induced IL-1s promote MMP-1 expression in an autocrine manner. This hypothesis is supported by the reduction of EDA-induced MMP-1 production by IL-1 receptor antagonist. The effect of EDA on MMP-1 production was reduced by connection with an adjacent type III repeat on either the NH2 or COOH side of EDA and was abolished by connection on both sides of EDA, suggesting that exposure of either the NH2 or COOH terminus of EDA domain by proteolytic cleavage releases the inducing activity. In agreement with these results, full-length cellular FN did not induce MMP-1 production. Furthermore, a 160-kDa EDA-positive FN fragment, which was purified from human placental tissue and corresponds to the region from NH2terminus through the EDA, induced MMP-1 production. Taken together, these results suggest that the EDA in FN fragments triggers alterations of cell physiology and plays a role in matrix degradation in joint connective tissue.

Angiopoietin-related growth factor (AGF) promotes epidermal proliferation, remodeling, and regeneration.

We report here the identification of an angiopoietin-related growth factor (AGF). To examine the biological function of AGF in vivo, we created transgenic mice expressing AGF in epidermal keratinocytes (K14-AGF). K14-AGF mice exhibited swollen and reddish ears, nose and eyelids. Histological analyses of K14-AGF mice revealed significantly thickened epidermis and a marked increase in proliferating epidermal cells as well as vascular cells in the skin compared with nontransgenic controls. In addition, we found rapid wound closure in the healing process and an unusual closure of holes punched in the ears of K14-AGF mice. Furthermore, we observed that AGF is expressed in platelets and mast cells, and detected at wounded skin, whereas there was no expression of AGF detected in normal skin tissues, suggesting that AGF derived from these infiltrated cells affects epidermal proliferation and thereby plays a role in the wound healing process. These findings demonstrate that biological functions of AGF in epidermal keratinocytes could lead to novel therapeutic strategies for wound care and epidermal regenerative medicine.

Mouse ULK2, a novel member of the UNC-51-like protein kinases: unique features of functional domains

The UNC-51 serine/threonine kinase of C. elegans plays an essential role in axonal elongation, and unc-51 mutants exhibit uncoordinated movements. We have previously identified mouse and human cDNAs encoding UNC-51-like kinase (ULK1). Here we report the identification and characterization of the second murine member of this kinase family, ULK2. Mouse ULK2 cDNA encodes a putative polypeptide of 1033 aa which has an overall 52% and 33% amino acid identity to ULK1 and UNC-51, respectively. ULKs and UNC-51 share a typical domain structure of an amino-terminal kinase domain, a central proline/serine rich (PS) domain, and a carboxy-terminal (C) domain. Northern blot analysis showed that ULK2 mRNA is widely expressed in adult tissues. In situ hybridization analysis indicated that ULK2 mRNA is ubiquitously localized in premature as well as mature neurons in developing nervous system. ULK2 gene was mapped to mouse chromosome 11B1.3 and rat chromosome 10q23 by FISH. HA-tagged ULK2 expressed in COS7 cells had an apparent molecular size of ∼150 kDa and was autophosphorylated in vitro. Truncation mutants suggested that the autophosphorylation occurs in the PS domain. Although expression of ULK2 failed to rescue unc-51 mutant of C. elegans, a series of ULK2/UNC-51 chimeric kinases revealed that function of the kinase and PS domains are conserved among species, while the C domain acts in a species-specific manner. These results suggest that ULK2 is involved in a previously uncharacterized signaling pathway in mammalian cells.

The gp116 of the gp58/116 complex of human cytomegalovirus represents the amino-terminal part of the precursor molecule and contains a neutralizing epitope

The glycoprotein complex gp58/116 of human cytomegalovirus (HCMV) represents a dominant antigen for the humoral immune response. We have used the human monoclonal antibody C23, which is capable of neutralizing HCMV in tissue culture without the addition of complement, to study the origin of gp116 as well as the amino acid sequence recognized by the antibody. Our results show that gp116 is derived from the same open reading frame as gp58 and that it represents the amino-terminal portion of the precursor protein. Using prokaryote-expressed beta-galactosidase-gp116 fusion proteins, the binding site of C23 was located to between amino acids 27 to 84 of the amino-terminal portion of gp116. Analyses of HCMV-positive human sera revealed that this portion of the molecule is immunogenic during natural infection.

ASB proteins interact with Cullin5 and Rbx2 to form E3 ubiquitin ligase complexes

The ankyrin repeat and SOCS box (ASB) family is composed of 18 proteins from ASB1 to ASB18 and belongs to the suppressor of cytokine signaling (SOCS) box protein superfamily. ASB2 was recently shown to interact with a certain Cul–Rbx module to form an E3 ubiquitin (Ub) ligase complex, but the functional composition of the ASB‐containing E3 Ub ligase complexes remains to be characterized. Here, we show that ASB proteins interact with Cul5–Rbx2 but neither Cul2 nor Rbx1 in cells. Mutational analysis revealed that the highly conserved amino acid sequences of the BC box and Cul5 box in the SOCS box of ASB proteins were essential for the interaction with Cul5–Rbx2. Although ASB proteins show slight divergences from the consensus sequences of the BC box and Cul5 box, all five tested ASB proteins bound to Cul5–Rbx2. Furthermore, all three tested ASB complexes containing Cul5–Rbx2 were found to have E3 Ub ligase activity. These findings suggest that the ASB family proteins interact with Cul5–Rbx2 to form E3 Ub ligases and play significant roles via a ubiquitination‐mediated pathway.

Human Monoclonal Antibodies Neutralizing Human Cytomegalovirus

Hybridomas producing human monoclonal antibodies (MAbs) against human cytomegalovirus (CMV) were generated by fusion of human spleen cells and mouse myeloma cells. Two of the six MAbs obtained neutralized viral infectivity even at concentrations lower than 1 microgram/ml. One MAb required complement for neutralization but the other did not. Both MAbs recognized viral proteins of Mr 130,000 and 55,000. Furthermore, these neutralizing MAbs bound to the surface membrane of CMV-infected cells. These results suggest that human MAbs may provide a new means of passive immunization against CMV infection in humans.

Immunoprotective Human Monoclonal Antibodies against Five Major Serotypes of Pseudomonas aeruginosa

Human monoclonal antibodies (Mabs) against the O antigens of Pseudomonas aeruginosa lipopolysaccharides (LPS) were produced by cell fusion between human tonsillar lymphocytes and P3-X63-Ag8-U1 (P3U1) mouse myeloma cells. To obtain human Mabs efficiently, 6 d culture supernatants of pokeweed-mitogen-stimulated lymphocytes (21 cultures from peripheral blood and 76 from tonsils) were assayed by ELISA. Five tonsillar lymphocytes which produced IgG antibody specific for P. aeruginosa LPS were preselected for fusion. The human Mabs, named P1-1 (IgG2, kappa), P5-1 (IgG2, lambda), P7-1 (IgG2, lambda), P8-1 (IgG2, lambda) and P10-1 (IgG2, kappa), bound with high specificity to Homma standard serotype strains A, E, B, G and I, respectively, and recognized O antigens. Each Mab showed opsonophagocytic killing activity of the corresponding serotype strain. Four of the Mabs caused agglutination at a very low concentration; a rather higher concentration of P7-1 was required for this effect. Although all the Mabs conferred type-specific protection against peritoneal infection, the strongly agglutinating Mabs provided better protection than the moderately agglutinating P7-1. The protective activity of P8-1 was estimated in compromised mice. A low dose (PD50 0.5-0.6 microgram per mouse) of P8-1 prevented subcutaneous infection in burned mice and peritoneal infection in leucopenic mice. All the hybridomas described here could be cultured in serum-free medium, and they have continued to secrete human Mabs for more than 14 months at rates of 10-20 micrograms per 10(6) cells in 24 h. These results suggested that these five human Mabs specific for O antigens might be useful in the prophylaxis and treatment of P. aeruginosa infections.