Nobutaka Wakamiya

Molecular cloning of a novel human collectin from liver (CL-L1).

Collectins are a C-lectin family with collagen-like sequences and carbohydrate recognition domains. These proteins can bind to carbohydrate antigens of microorganisms and inhibit their infection by direct neutralization and agglutination, the activation of complement through the lectin pathway, and opsonization by collectin receptors. Here we report the cloning of a cDNA encoding human collectin from liver (CL-L1 (collectin liver 1)) that has typical collectin structural characteristics, consisting of an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain, and a carbohydrate recognition domain. The cDNA has an insert of 831 base pairs coding for a protein of 277 amino acid residues. The deduced amino acid sequence shows that this collectin has a unique repeat of four lysine residues in its C-terminal area. Northern blot, Western blot, and reverse transcription-polymerase chain reaction analyses showed that CL-L1 is present mainly in liver as a cytosolic protein and at low levels in placenta. More sensitive analyses by reverse transcription-polymerase chain reactions showed that most tissues (except skeletal muscle) have CL-L1 mRNA. Zoo-blot analysis indicated that CL-L1 is limited to mammals and birds. A chromosomal localization study indicated that the CL-L1 gene localizes to chromosome 8q23-q24.1, different from chromosome 10 of other human collectin genes. Expression studies of fusion proteins lacking the collagen and N-terminal domains produced in Escherichia coli affirmed that CL-L1 binds mannose weakly. CL-L1 and recombinant CL-L1 fusion proteins do not bind to mannan columns. Analysis of the phylogenetic tree of CL-L1 and other collectins indicated that CL-L1 belongs to a fourth subfamily of collectins following the mannan-binding protein, surfactant protein A, and surfactant protein D subfamilies including bovine conglutinin and collectin-43 (CL-43). These findings indicate that CL-L1 may be involved in different biological functions.

Identification and Characterization of a Novel Human Collectin CL‐K1

Collectins are a family of C‐type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host‐defense. Here we report the cloning and characterization of a novel collectin CL‐K1. RT‐PCR analyses showed CL‐K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL‐K1 cDNA expressing CHO cells revealed that CL‐K1 is expressed as a secreted protein. CL‐K1 is found in blood by immunoblotting and partial amino acid analyses. CL‐K1 showed Ca2+‐dependent sugar binding activity of fucose and weakly mannose but not N‐acetyl‐galactosamine, N‐acetyl‐glucosamine, or maltose, though mannose‐binding lectin (MBL) containing similar amino acid motif. CL‐K1 can recognize specially several bacterial saccharides due to specific sugar‐binding character. Elucidation of the role of two ancestor collectins of CL‐K1 and CL‐L1 could lead to see the biological function of collectin family.

Mannose-binding lectin gene: polymorphisms in Japanese patients with systemic lupus erythematosus, rheumatoid arthritis and Sjögren's syndrome.

Mannose-binding lectin (MBL) is a key element of the innate immunity, with a structure similar to complement C1q. Serum MBL levels are greatly affected by the polymorphisms of the MBL gene. In particular, codon 54 mutation of the MBL gene results in a significant reduction of serum MBL. To determine whether polymorphism of the MBL gene is associated with occurrence of systemic lupus erythematosus (SLE), rheumatoid arthritis and Sjögren’s syndrome in the Japanese population, we analyzed the MBL gene polymophisms of these patients and controls, by polymerase chain reaction-restriction fragment length polymorphism methods. We found that patients studied had a significantly higher frequency of having homozygous codon 54 mutation compared to controls. In particular, patients with SLE or Sjögren’s syndrome showed higher probabilities of being homozygous for this mutation. Among subjects with the same genotype, SLE patients tended to have higher serum MBL concentration than controls. Analysis of the promotor region suggested that SLE patients heterozygous for the codon 54 mutation have a higher probability of having a low producing haplotype for the gene without the codon 54 mutation. We conclude that persons homozygous for codon 54 mutation of the MBL gene may be prone to occurrence of autoimmune disorders including SLE, in the Japanese. MBL may have protective effects on occurrence and progression of SLE.

HUMAN MANNAN-BINDING LECTIN INHIBITS THE INFECTION OF INFLUENZA A VIRUS WITHOUT COMPLEMENT

Mannan‐binding lectin (MBL) is a C‐type serum lectin that is believed to play an important role in innate immunity. It is one of the collectin family, which is characterized by having a collagen‐like sequence and a carbohydrate recognition domain. MBL can bind to sugar determinants of several micro‐organisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Bovine conglutinin and mouse MBL inhibit the infective and haemagglutinating activities of influenza A viruses. To identify the direct antiviral activity of human MBL against influenza A viruses that does not depend on complement activation or opsonization, we isolated native MBL from human serum and produced a recombinant MBL in Chinese hamster ovary (CHO) cells using a pNOW/CMV‐A expression vector system. Native and recombinant human MBL exhibited neutralization activity against A/Ibaraki/1/90 (H3N2), with the plaque focus reduction assay at the viral attachment phase. Their activities were inhibited by EDTA, mannose and anti‐human MBL antibody. Furthermore, at the viral expansion phase both MBL in culture medium prevented viral spreading from primary infected cells to neighbour cells. A virus recovery study using EDTA indicated that interaction between MBL and virus was reversible and non‐damaging to the virus. Lectin blot and immunohistochemistry assays showed that these antiviral activities involved binding between MBL and two viral envelope proteins, haemagglutinin and neuraminidase. These findings suggest that human MBL can play an important role in innate immunity by direct viral neutralization and inhibition of viral spread, as well as an indirect role through opsonization and complement activation.

Mannose-binding lectin polymorphisms in patients with hepatitis C virus infection.

BACKGROUND Persistent infection with hepatitis C virus (HCV) leads to liver cirrhosis (LC) and often to liver cancer. Little is known about host factors that determine the variable natural history. Mannose-binding lectin (MBL) is an important constituent of the innate immune system. In white patients there is an association between codon 52 mutation of the MBL gene and persistent hepatitis B virus (HBV) infection. To determine whether MBL gene polymorphisms affect the course of HCV infection, we investigated the association between MBL gene polymorphisms and HCV infection in Japanese subjects. METHODS Fifty-two HCV-infected Japanese patients (8 with chronic inactive hepatitis (CIH), 31 with chronic active hepatitis (CAH), 13 with LC) and 50 normal controls were studied. MBL gene mutations were determined by means of polymerase chain reaction and restriction fragment length polymorphism analyses. RESULTS Codon 52 and codon 57 mutations were absent in all subjects. Homozygous mutation in codon 54 was present in one (0.9%) patient. Heterozygous codon 54 mutation was present in 17 (32%) of the 52 patients and in 21 (41%) of the controls. No significant difference in the frequency of codon 54 mutation was observed between patient and control groups. However, although no significant relationship was observed between MBL polymorphisms and the levels of HCV RNA, all patients with heterozygous or homozygous codon 54 mutations had CAH or LC. In contrast, 8 of the 34 patients without codon 54 mutation remained at CIH. (P = 0.0405). CONCLUSION MBL may be one of the factors that influence the course of HCV infection.Background: Persistent infection with hepatitis C virus (HCV) leads to liver cirrhosis (LC) and often to liver cancer. Little is known about host factors that determine the variable natural history. Mannose-binding lectin (MBL) is an important constituent of the innate immune system. In white patients there is an association between codon 52 mutation of the MBL gene and persistent hepatitis B virus (HBV) infection. To determine whether MBL gene polymorphisms affect the course of HCV infection, we investigated the association between MBL gene polymorphisms and HCV infection in Japanese subjects. Methods: Fifty-two HCV-infected Japanese patients (8 with chronic inactive hepatitis (CIH), 31 with chronic active hepatitis (CAH), 13 with LC) and 50 normal controls were studied. MBL gene mutations were determined by means of polymerase chain reaction and restriction fragment length polymorphism analyses. Results: Codon 52 and codon 57 mutations were absent in all subjects. Homozygous mutation in codon 54 was present in one (0.9%) patient. Heterozygous codon 54 mutation was present in 17 (32%) of the 52 patients and in 21 (41%) of the controls. No significant difference in the frequency of codon 54 mutation was observed between patient and control groups. However, although no significant relationship was observed between MBL polymorphisms and the levels of HCV RNA, all patients with heterozygous or homozygous codon 54 mutations had CAH or LC. In contrast, 8 of the 34 patients without codon 54 mutation remained at CIH. (P = 0.0405). Conclusion: MBL may be one of the factors that influence the course of HCV infection.

Recombinant expression of human mannan-binding lectin

Mannan-binding lectin (MBL) constitutes an important part of the innate immune defence by effecting the deposition of complement on microbial surfaces. MBL deficiency is among the most common primary immunodeficiencies and is associated with recurrent infections and symptoms of poor immune complex clearance. Plasma-derived MBL has been used in reconstitution therapy but concerns over viral contamination and production capacity point to recombinant MBL (rMBL) as a future source of this protein for clinical use. Natural human MBL is an oligomer of up to 18 identical polypeptide chains. The synthesis of rMBL has been accomplished in several mammalian cell lines, however, the recombinant protein differed structurally from natural MBL. In this, study we compare rMBL produced in myeloma cells, Chinese hamster ovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK) cells. We report that rMBL structurally and functionally similar to natural MBL can be obtained through synthesis in the human embryonic kidney cells followed by selective carbohydrate affinity chromatography.

Two chicken monoclonal antibodies specific for heterophil Hanganutziu-Deicher antigens.

Two chicken monoclonal antibodies (MAbs), HU/Ch2-7 and HU/Ch6-1, against heterophil Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (NeuGc) at a terminal carbohydrate were established by cell fusions using chicken B cell lines lacking thymidine kinase and spleen cells from chickens immunized with II3NeuGc alpha-LacCer (HD3). The reactivities of these MAbs against several gangliosides including NeuGc-containing glycosphingolipids were examined by a thin-layer chromatography/immunostaining method. MAb HU/Ch2-7 (IgG) reacted strongly with HD3 and IV3NeuGc alpha-nLc4Cer (HD5) and weakly with VI3NeuGc alpha-nLc6Cer (HD7) and 4-O-acetyl-HD3. HU/Ch6-1 (IgG) reacted with HD3 and HD5, but did not react with the other HD antigens. The reactivities of these MAbs against HD antigen were greatly reduced by pre-treatment of the antigen with neuraminidase. These MAbs did not react with N-acetylneuraminic acid-containing gangliosides (GM1 and GM3). These results indicate that these two chicken MAbs are directed toward the antigenic epitope containing the NeuGc.

Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins

The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa-3) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg0) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa-3 and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa-3 determined the binding affinity for HSP47. Maximal binding was observed when Yaa-3 was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr-3 and one Arg0. The results revealed that HSP47 recognizes the Yaa-3 and Arg0 residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.

Scavenger Receptor Collectin Placenta 1 (CL-P1) Predominantly Mediates Zymosan Phagocytosis by Human Vascular Endothelial Cells

Collectin placenta 1 (CL-P1), a recently discovered scavenger receptor, mediates the uptake of oxidized low density lipoprotein and microbes. In this study, we investigated CL-P1-mediated binding and ingestion of yeast-derived zymosan bioparticles using Chinese hamster ovary (CHO) cells stably expressing human CL-P1 (CHO/CL-P1) and human vascular endothelial cells constitutively expressed CL-P1. The uptake of zymosan by CHO/CL-P1 was dependent upon the level of CL-P1 expressed on the membrane and was inhibited by cytochalasin D and wortmannin. The binding of zymosan was also inhibited by ligands of other scavenger receptors such as poly(I) and dextran sulfate. Real time reverse transcription-PCR analyses showed that other scavenger receptors, namely LOX-1, Stabilin-2, or macrophage receptor with collagenous structure (MARCO), were not expressed in human umbilical vein endothelial cells isolated from different individuals. Nonopsonic zymosan ingestion was inhibited in three primary cultured vascular endothelial cells, including different human umbilical vein endothelial cells from nine individuals treated with CL-P1 small interfering RNAs, although small interfering RNAs of other scavenger receptors had no effect on zymosan uptake in these cells. Furthermore, we confirmed that CL-P1 is expressed in human and murine vascular endothelial layers. Our results demonstrated that CL-P1 predominantly mediated phagocytosis for fungi in vascular endothelia.

Isolation and characterization of conglutinin as an influenza A virus inhibitor.

Normal horse and guinea pig sera contain alpha 2-macroglobulin which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. On the other hand, normal bovine serum contains a component termed beta inhibitor that inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H1 and H3 subtypes. To investigate the nature of the beta inhibitor of influenza A virus, we purified the conglutinin and examined its characteristics. First, we found a high correlation between the hemagglutination inhibition(HI) titer and conglutinin titer in several bovine sera (r = 0.906, p less than 0.005). The HI of bovine serum was mainly dependent on conglutinin because the HI activity was abrogated by N-acetylglucosamine but not by D-mannose. The conglutinin, purified from bovine serum, had neutralizing-activity as well as HI activity on influenza A viruses of the H1 and H3 subtypes. The HI activity of conglutinin was heat stable (56 degrees C, 30 min), Ca(++)-dependent, and resistant to both neuraminidase and periodate treatments. The HI activity of purified conglutinin was blocked by N-acetylglucosamine but not by D-mannose. The conglutinin was bound to hemagglutinin which had high mannose and complex sugar chains and its binding was inhibited by N-acetylglucosamine and dependent on divalent cations. These data indicate that the beta-like inhibitor activity of bovine serum is mainly dependent on conglutinin which inhibits hemagglutination and neutralizes the virus infectivity by its binding to a carbohydrate site at the HA.