Synthesis and characterization of a recombinant myohemerythrin protein encoded by a synthetic gene.

The antigenic epitopes of the myohemerythrin (MHr) molecule have been studied extensively. The critical amino acid residues responsible for its immune recognition have been identified by using synthetic peptides and the technique of epitope scanning. To assess the true relevance of these techniques for determining the molecular mechanism of antigenic recognition and immunogenicity, the results obtained with isolated peptides should be tested in the context of the folded protein. To this end, we have designed and constructed a synthetic MHr gene, in modular form, which will allow subsequent alterations of nucleotide sequence encoding epitopes of interest. We have produced the recombinant protein at high level, and have shown by several criteria that it possesses the chemical, physical and immunological properties of the native worm protein. Thus, we have developed a valuable system for detailed immunological studies of the structure and chemistry required for antibody binding to protein.

Antibacterial properties of hemerythrin of the sand worm Nereis diversicolor.

OBJECTIVES: To investigate the immune defense of the annelid Nereis diversicolor and the key role of a oxygen-binding protein, the metalloprotein MPII animals were subjected to bacteria infection. METHODS AND RESULTS: Using RACE-PCR, we have cloned the complete cDNA coding for the MPII related to the hemerythrin family in the sand worm Hediste diversicolor. This cDNA (883 pb) codes for a polypeptide of 119 amino acid residues with no signal peptide. Previous works have identified this protein as a cadmium scavenger. We here clearly demonstrated that this protein is also involved in the worm defence towards bacteria growth by its iron scavenger ability. This protein is expressed and produced in a haematopoietic center that floats freely in the coelomic fluid before stored in a particular hemocyte type: the granulocyte type 1. During bacterial challenge, this protein contained in these cells is discharged into the blood stream 3-4 hours after the infection and remains active for approximately 10 hours. This time period blocks progression of the pathogen and its attachment to tissues. CONCLUSION: These results reflect that MPII in conjunction with others partners like lysozyme act as defence molecule for the sand worm.

Alternate protein frameworks for molecular recognition.

In an effort to determine whether proteins with structures other than the immunoglobulin fold can be used to mimic the ligand binding properties of antibodies, we generated a library from the four-helix bundle protein cytochrome b562 in which the two loops were randomized. Panning of this library against the bovine serum albumin (BSA) conjugate of N-methyl-p-nitrobenzylamine derivative 1 by phage display methods yielded cytochromes in which residues Trp-20, Arg-21, and Ser-22 in loop A and Arg-83 and Trp-84 in loop B were conserved. The individual mutants, which fold into native-like structure, bind selectively to the BSA-1 conjugate with micromolar dissociation constants (Kd), in comparison to a monoclonal antibody that binds selectively to 1 with a Kd of 290 nM. These and other antibody-like receptors may prove useful as therapeutic agents or as reagents for both intra- and extracellular studies.