J. Am. Chem. Soc. 2009, 131, 13192-13193
DOI: 10.1021/ja903631h Read in JACS
Highly fluorinated amino acids have been used to stabilize helical proteins for potential application in various protein-based biotechnologies. However, many proteins used for therapeutics and biosensors involve β-sheet proteins such as antibodies. Accordingly, we explored the effect of several highly fluorinated amino acids on β-sheet stability including (S)-2-amino-4,4,4-trifluorobutyric acid (Atb), (S)-5,5,5′,5′-tetrafluoroleucine (Qfl), (S)-5,5,5,5′,5′,5′-hexafluoroleucine (Hfl), and (S)-pentafluorophenylalanine (Pff). Nine proteins based on the protein G B1 domain I6A T44A mutant (GB1) with various amino acids at the solvent exposed guest position 53 in the internal strand 4 were synthesized, purified, and investigated by thermal denaturation monitored by circular dichroism spectroscopy. Based on the thermal denaturation data, GB1 stability is affected by the amino acid at the guest position 53. Apparently, introducing fluorine results in more stable GB1 mutants (Pff > Phe, Hfl > Qfl > Leu, Atb > Abu). In particular, GB1 becomes more stable upon introducing fluorines by up to 0.35 kcal·mol−1·residue−1. Overall, these results suggest that fluoro-amino acids may be worthwhile building blocks to explore for stabilizing β-sheet proteins, which are especially important for biotechnologies such as protein therapeutics and biosensors.
Neoplasia 2009, 11, 780-792
PMCID: PMC2713588 Read in Neoplasia
The tumor-associated carbohydrate Thomsen-Friedenreich antigen (TF-Ag; Galβ1-3GalNAcα-O-Ser/Thr) is overexpressed on the cell surface of several types of tumor cells, contributing to cancer cell adhesion and metastasis to sites containing TF-Ag-binding lectins. A highly specific immunoglobulin G3 monoclonal antibody (Ab) developed to TF-Ag (JAA-F11) impedes TF-Ag binding to vascular endothelium, blocking a primary metastatic step and providing a survival advantage. In addition, in patients, even low levels of antibodies to TF-Ag seem to improve prognosis; thus, it is expected that vaccines generating antibodies toward TF-Ag would be clinically valuable. Unfortunately, vaccinations with protein conjugates of carbohydrate tumor-associated Ags have induced clinically inadequate immune responses. However, immunization using peptides that mimic carbohydrate Ags such as Lewis has resulted in both Ab and T-cell responses. Here, we tested the hypothesis that vaccinations with unique TF-Ag peptide mimics may generate immune responses to TF-Ag epitopes on tumor cells, useful for active immunotherapy against relevant cancers. Peptide mimics of TF-Ag were selected by phage display biopanning using JAA-F11 and rabbit anti-TF-Ag Ab and were analyzed in vitro to confirm TF-Ag peptide mimicry. In vitro, TF-Ag peptide mimics bound to TF-Ag-specific peanut agglutinin and blocked TF-Ag-mediated rolling and stable adhesion of cancer cells to vascular endothelium. In vivo, the immunization with TF-Ag-mimicking multiple antigenic peptides induced TF-Ag-reactive Ab production. We propose that this novel active immunotherapy approach could decrease tumor burden in cancer patients by specifically targeting TF-Ag-positive cancer cells and blocking metastasis.