Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.

Stability of specific immunoglobulin secretion by EBV-transformed lymphoblastoid cells and human-murine heterohybridomas

The purpose of this project was to determine if stability of specific antibody secretion improved after fusion of Epstein-Barr virus (EBV)-transformed lymphoblastoid cells with P3X63Ag8.653 murine myeloma cells. Production of human monoclonal antibodies by Epstein-Barr virus transformation and somatic cell fusion has been used by many laboratories, however the steps involved have not been fully optimized. B lymphocytes isolated from the peripheral blood of normal donors were enriched for Thomsen-Friedenreich (T) antigen-reactive cells by panning on asialoglycophorin. The EBV-transformed lymphoblastoid cell lines generated from asialoglycophorin-adherent B lymphocytes were treated in three different manners: (1) cloned and maintained in culture as monoclonal lymphoblastoid cell lines, (2) cloned and fused with murine myeloma cells or (3) fused shortly after transfomation without prior cloning. Cloned lymphoblastoid cell lines maintained in culture without fusion either died or lost specific antibody secretion within five months. Uncloned lymphoblastoid cells remained viable for up to three months but lost specific antibody secretion within two months probably due to overgrowth by nonspecific clones. In an attempt to increase longevity and to stabilize specific antibody secretion by these cells, the cloned lymphoblastoid cells were fused with murine myeloma cells. In nine of ten fusions no hybrids were recovered. As an alternate approach, uncloned lymphoblastoid cells secreting T antigen-specific antibody were hybridized with murine myeloma cells, hybrids secreting T antigen-specific antibody were recovered in six of seven fusions. Furthermore, T antigen-specific antibodies of high titer were secreted by the heterohybridoma clones for more than five months of continuous culture. These heterohybridoma cells secreted more immunoglobulin, produced greater titers of antibody and maintained specific antibody secretion longer than either monoclonal or polyclonal EBV-transformed lymphoblastoid cells. These studies have conclusively demonstrated that fusion of polyclonal lymphoblastoid cells secreting T antigen-specific antibody with murine myeloma cells results in prolongation of human monoclonal antibody production compared with unfused monoclonal or polyclonal lymphoblastoid cell lines. This procedure should be generally applicable for the production of stable human monoclonal antibody-secreting cells lines from peripheral blood lymphocytes. ^

Variables affecting the stability of multiple cloning sites and hairpin structures in retroviral vectors

Retroviruses are RNA viruses that replicate through a double-stranded DNA intermediate. The viral enzyme reverse transcriptase copies the retroviral genomic RNA into this DNA intermediate through the process of reverse transcription. Many variables can affect the fidelity of reverse transcriptase during reverse transcription, including specific sequences within the retroviral genome. ^ Previous studies have observed that multiple cloning sites (MCS) and sequences predicted to form stable hairpin structures are hotspots for deletion during retroviral replication. The studies described in this dissertation were performed to elucidate the variables that affect the stability of MCS and hairpin structures in retroviral vectors. Two series of retroviral vectors were constructed and characterized in these studies. ^ Spleen necrosis virus-based vectors were constructed containing separate MCS insertions of varying length, orientation, and symmetry. The only MCS that was a hotspot for deletion formed a stable hairpin structure. Upon more detailed study, the MCS previously reported as a hotspot for deletion was found to contain a tandem linker insertion that formed a hairpin structure. Murine leukemia virus-based vectors were constructed containing separate sequence insertions of either inverted repeat symmetry (122IR) that could form a hairpin structure, or little symmetry (122c) that would form a less stable structure. These insertions were made into either the neomycin resistance marker ( neo) or the hygromycin resistance marker (hyg) of the vector. 122c was stable in both neo and hyg, while 122IR was preferentially deleted in neo and was remarkably unstable in hyg. ^ These results suggest that MCS are hotspots for deletion in retroviral vectors if they can form hairpin structures, and that hairpin structures can be highly unstable at certain locations in retroviral vectors. This information may contribute to improved design of retroviral vectors for such uses as human gene therapy, and will contribute to a greater understanding of the basic science of retroviral reverse transcription. ^