Search

The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan-binding lectin (MBL) was investigated by solid-phase binding assays. Calreticulin showed saturable and time-dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non-covalent and biphasic with an initial salt-sensitive phase followed by a more stable salt-insensitive interaction. For plasma-derived MBL, known to be complexed with MBL-associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP-2, MASP-3 and MAp19, but not by the MASP-2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co-receptor/chaperone for both proteins. In conclusion, the potential MBL co-receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.

Mannan-binding lectin (MBL), a human plasma protein, plays an important role in the innate immune defence. MBL recognizes microorganisms through surface carbohydrate structures. Due to genetic polymorphisms, MBL plasma concentrations range from 5 to 10,000 ng/mL. Approximately 30% of the human population have low levels of MBL (below 500 ng/mL). MBL deficiency is associated with increased susceptibility to infections in immunosuppressed individuals, e.g., during chemotherapeutically induced neutropenia. Replacement therapy with MBL may be beneficial in this patient group, and recombinant human MBL (rhMBL) is in development as a novel therapeutic approach. To assess the safety, tolerability, and pharmacokinetics of rhMBL, a placebo-controlled double-blinded study was performed in MBL-deficient healthy male subjects. rhMBL was administered as both single intravenous (i.v.) infusions (0.01, 0.05, 0.1, and 0.5 mg/kg) and repeated i.v. infusions (0.1 or 0.3 mg/kg given at 3-day intervals). There were no difference in incidence and type of adverse events reported in the study between the groups of subjects receiving rhMBL and the placebo group. All adverse events reported as drug-related were mild and no serious adverse events were recorded. There were no clinically significant changes in laboratory evaluations, ECG or vital signs, and no anti-MBL antibodies were detected following rhMBL administration. After single i.v. doses of rhMBL the maximal plasma levels increased in a dose-dependent manner reaching a geometric mean of 9710 ng/mL+/-10.5% in the highest dose group (0.5 mg/kg), with an elimination half-life of approximately 30 h. No rhMBL accumulation in plasma was observed following repeat dosing. Administration of rhMBL restored the ability to activate the MBL pathway of the complement system without non-specific activation of the complement cascade. In conclusion, no safety or tolerability concern was raised following rhMBL administration no signs of immunogenicity detected, and an rhMBL plasma level judged sufficient to achieve therapeutic benefit (>1000 ng/mL) can be achieved.

A perfused cell-culture process was developed to investigate the stability of IRF-1-mediated proliferation control in BHK cells and to evaluate the efficacy of a novel promoter in these cells. The cell density of proliferation-controlled producer cells was effectively regulated for over 7 weeks in a microcarrier-based continuously perfused bioreactor. An IRF-1-inducible promoter was employed to express a heterodimeric IgG antibody as a relevant model protein. Basal expression levels were equivalent to that of a highly active viral promoter, while productivity increased up to sixfold during growth arrest. However, no stably expressing clone was isolated in this study. Protein expression decreased gradually with time and could not be induced further in subsequent growth-repression cycles. The results demonstrate that the regulatory system is sufficiently stable to allow controlled growth in a continuous scalable reactor system and that productivity increases can be achieved in a proliferation controlled microcarrier culture., (Copyright 2000 John Wiley & Sons, Inc.)

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of (35)S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVIII producing cells, could be responsible for the cleavage of rFVIII. Among several protease inhibitors tested, only bacitracin was found to diminish the extent of cleavage. Phosphatidylserine also protected rFVIII against cleavage, probably by binding to rFVIII.