Katsuki Ohtani

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.

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.

BACKGROUNDnPersistent 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.nnnMETHODSnFifty-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.nnnRESULTSnCodon 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).nnnCONCLUSIONnMBL 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.

High-level and effective production of human mannan-binding lectin (MBL) in Chinese hamster ovary (CHO) cells.

We have developed a high-expression system of recombinant human mannan-binding lectin (MBL) with CHO cells. Geneticin-resistant transformants harboring human MBL cDNA in the expression vector pNOW/CMV-A were screened by immunoblot analysis for secretion of recombinant MBL. Cloning and selection by both geneticin and methotrexate resulted in the production of recombinant MBL to a final concentration of 128.8 microg/ml in media after four days of culture. SDS-PAGE and gel-filtration analyses showed that recombinant MBL is characterized by two lower-order oligomeric structures (apparent molecular weights: 1150 kDa and 300 kDa) compared to native MBL (apparent molecular weight: 1300 kDa). The recombinant human MBL has both sugar-binding and complement activation activity and, like native MBL, can inhibit hemagglutination of influenza A virus. Lectin blots with recombinant MBL indicate that it can bind such microorganisms as HIV and influenza virus suggesting that it might inhibit their infection of hosts. This high-level expression of human MBL with the full range of biological activity will be useful for studies on the immunological role of MBL in humans.