Overproduction of recombinant human mannose-binding lectin (MBL) in Chinese hamster ovary cells.

Mannose-binding lectin (MBL) is an important serum protein that functions in the innate immune system and has been considered to have therapeutic potential in MBL replacement therapies for patients with deficient or low levels of MBL. In this study, we established a Chinese hamster ovary (CHO) cell line that overexpresses the recombinant human MBL (rhMBL) protein. In an 11-day batch culture process using a 30-L bioreactor (20-L working volume) and serum-free medium, these cells could produce over 226 mg/L of rhMBL protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a three-step chromatographic procedure that resulted in a recovery rate of approximately 55%. This purified rhMBL protein adopted oligomeric bouquet-like structures that were similar to those of native MBL present in human blood, and these oligomeric structures were reported to be critical in MBL functions. We further demonstrated in carbohydrate binding and complementation activation assays that this rhMBL protein was functionally active with very similar dissociation constants and half maximal effective concentrations to those of native MBL.

Analysis of mannose-binding lectin 2 (MBL2) genotype and the serum protein levels in the Korean population.

Mannose-binding lectin (MBL) is a C-type lectin produced by the liver and involved in the innate immune response. We have analyzed six SNPs of MBL2 gene--three at promoter (-550, -435, and -221), one at 5'-untranslational region (UTR) (+4), and two at coding (Gly54Asp and Leu126Leu) regions--in the Korean population (N=129), and have correlated genotypes with the serum concentration and functional characteristics. Of those, the Asp54 allele (P<10(-15)), L allele at -550 (P<10(-7)), and P allele at +4 (P=0.012) were correlated with low MBL levels. The effect of the X allele at -221 on MBL levels in the Korean population appeared to be less profound than that of other populations. The highest MBL producing promoter haplotype in the Korean population was HYP, followed by LYQ and LYP, and then LXP. From functional analysis of MBL, low MBL levels were correlated with low mannan-binding, low C4 complement activation, and lack of high ordered oligomers. Our results support that the promoter and coding polymorphisms of MBL are correlated with its functional activity as well as circulating levels, and the association patterns are quite similar to those of other populations.

Use of pre-S protein-containing hepatitis B virus surface antigens and a powerful adjuvant to develop an immune therapy for chronic hepatitis B virus infection.

A hepatitis B virus (HBV) vaccine has been developed using a new adjuvant and HBV surface antigens produced from a CHO cell line. The purified HBV surface antigens are composed of L protein, M protein, and S protein in a mixture of 20- and 40-nm-diameter particles and filamentous forms. This HBV surface antigen, formulated with L-pampo, a proprietary adjuvant, induced 10 times more antibody than the same antigen with alum and was capable of inducing strong immune responses in three different HBV transgenic mice. In spite of the presence of a large amount of HBV antigen in the blood, no antibody against HBV surface antigen was normally detected in these transgenic mice. After immunization, the HBV antigen was also cleared from the blood.

Mannose-binding lectin without the aid of its associated serine proteases alters lipopolysaccharide-mediated cytokine/chemokine secretion from human endothelial cells.

Coupling between certain pathogen-associated molecular patterns and corresponding pattern recognition receptors of endothelial cells is important for the mediation of vascular inflammatory responses. Mannose-binding lectin (MBL) recognizes certain carbohydrate structures of microbes and subsequently activates the complement system as well as facilitates the phagocytosis of targets. We investigated whether MBL can intervene in the interaction between bacterial lipopolysaccharide (LPS) and endothelial cells to modulate subsequent inflammatory responses. In response to LPS, human umbilical vein endothelial cells (HUVEC) produced various cytokines/chemokines. Addition of the purified human MBL/MBL-associated serine proteases (MASP) complex or recombinant human MBL enhanced LPS-mediated cytokine/chemokine secretion by HUVEC, including interleukin-8 (IL-8), IL-6 and monocyte chemoattractant protein-1 in a dose-dependent manner. This enhancing effect was ameliorated by the addition of anti-MBL antibody or mannan. Among the cytokines/chemokines we analysed, IL-6 showed the greatest increase of secretion in the presence of native MBL/MASP complex or recombinant MBL. MBL, regardless of its association with MASP, alters LPS-mediated cytokine/chemokine secretion of HUVEC. Besides the well-known functions of MBL, to activate the lectin-complement pathway and to facilitate clearance of targets, alteration of cytokine/chemokine secretion may provide an additional role for MBL in modulating vascular inflammation.

The influence of N-glycosylation and C-terminal sequence on secretion of HBV large surface antigen from S. cerevisiae.

In Saccharomyces cerevisiae, we synthesized and secreted L-HBVsAg (named as pre-S(Met1 to Asn174)::S(Met175 to Ile400)) and three mutants, i.e., pre-S degree degree::S (Asn15Gln and Asn123Gln), pre-S degree degree::S degree (Asn15Gln, Asn123Gln, and Asn320Gln), and pre-S degree degree::S degree degree (Asn15Gln, Asn123Gln, Asn233Gln, and Asn320Gln). All of the secreted pre-S::S was N-glycosylated, i.e., hyper-mannosylated. In the secretion of pre-S degree degree::S and pre-S degree degree::S degree, besides the hyper-mannosylated form, another immunoreactive protein with much lower molecular mass was observed, which seems to be unglycosylated form of pre-S degree degree::S and pre-S degree degree::S degree. Only a part of the secreted pre-S degree degree::S or pre-S degree degree::S degree molecules was N-glycosylated, and the site for the partial N-glycosylation seems to be Asn233 in S-antigen region. Compared to the N-glycosylated pre-S degree degree::S and pre-S degree degree::S degree, pre-S degree degree::S degree degree (non-N-glycosylated mutant) was secreted with lower secretion efficiency but showed apparent immunoreactivity to anti-S antigen monoclonal Ab. Interestingly, unlike pre-S degree degree::S degree degree with authentic C-terminus, the recombinant pre-S degree degree::S degree degree with C-terminal myc or poly-histidine tag (pre-S degree degree::S degree degree::tag) was almost all aggregated into insoluble proteins in the intracellular region. Conclusively, the C-terminal sequence and glycosylation in S-antigen region seem to be of crucial importance in determining the secretion efficiency of L-HBVsAg in S. cerevisiae.

The influence of glycosylation on secretion, stability, and immunogenicity of recombinant HBV pre-S antigen synthesized in Saccharomyces cerevisiae.

Three types of recombinant pre-S antigens (i.e., pre-S1S2) of hepatitis B virus (HBV) were synthesized in Saccharomyces cerevisiae and secreted into extracellular medium: wild type (pre-S1S2) and two mutant antigens, pre-S1 degrees S2 (Asn15Gln) and pre-S1 degrees S2 degrees (Asn15Gln and Asn123Gln). An N-terminus sequence (Ser5-Ala28) of human interleukin 1 beta (hIL-1 beta) was used as synthetic prosequence of recombinant HBV surface antigen (pre-S), secreted from S. cerevisiae. The expression cassette comprised the signal peptide of the killer toxin of Kluyveromyces lactis, the synthetic prosequence above, KEX2 dibasic endopeptidase cleavage site (-Lys-Arg-), and the surface antigen. The recombinant pre-S1S2 and pre-S1 degrees S2 were secreted in the hyper-mannosylated form, while the recombinant pre-S1 degrees S2 degrees was produced without N-glycosylation. It has been demonstrated that the two particular N-linked glycans at Asn15 and Asn123 interfered with the B-cell response to the HBV-derived pre-S1S2, resulting in low titers of pre-S1S2-neutralizing antibodies. This problem was overcome by eliminating both of the N-glycosylation signals. Despite enhanced immunogenicity, the recombinant pre-S1 degrees S2 degrees showed two major problems: (1) inefficient Kex2 cleavage process in the secretory pathway and (2) the severe proteolytic degradation by yeast proteases. The efficiency of Kex2 cleavage increased dramatically by removing N-glycosylation signal in the synthetic prosequence, but the proteolysis of pre-S1 degrees S2 degrees was somewhat inevitable. Further systematic approaches including modulation of degree of N-glycosylation or relocation of N-glycosylation sites in the recombinant pre-S1S2 may make it possible to achieve both enhanced immunogenicity and resistance towards proteolytic degradation of the secreted pre-S antigen.