Preferential rabbit antibody responses to C-termini of NOTCH3 peptide immunogens.

Antibodies raised in peptide-immunized rabbits have been used in biological research for decades. Although there has been wide implementation of this approach, specific proteins are occasionally difficult to target for multiple reasons. One consideration that was noted in mice is that humoral responses may preferentially target the carboxyl terminus of the peptide sequence which is not present in the intact protein. To shed light on the frequency of preferential rabbit antibody responses to C-termini of peptide immunogens, we present our experience with generation of rabbit antibodies to human NOTCH3. A total of 23 antibodies were raised against 10 peptide sequences of human NOTCH3. Over 70% (16 of 23) of these polyclonal antibodies were determined to be C-terminal preferring: NOTCH3 peptide-reactive antibodies largely targeted the terminating free carboxyl group of the immunizing peptide. The antibodies that preferred C-terminal epitopes reacted weakly or not at all with recombinant target sequences with extension the C-terminus that eliminated the free carboxyl group of the immunogen structure; furthermore, each of these antisera revealed no antibody reactivity to proteins truncated before the C-terminus of the immunogen. In immunocytochemical applications of these anti-peptide antibodies, we similarly found reactivity to recombinant targets that best binding to cells expressing the free C-terminus of the immunizing sequence. In aggregate, our experience demonstrates a strong propensity for rabbits to mount antibody responses to C-terminal epitopes of NOTCH3-derived peptides which is predicted to limit their use against the native protein. We discuss some potential approaches to overcome this bias that could improve the efficiency of generation of antibodies in this commonly utilized experimental paradigm.

Tripartite factors leading to molecular divergence between human and murine smooth muscle.

A large number of pre-clinical and developmental investigations involve experimental vertebrate animals, of which mice have emerged as a favored organism. Recognition of the differences between humans and mice is essential for assessment of the relevance of animal studies to humans. The primary purpose of this study was to gauge the conservation between human and mouse vascular smooth muscle cell (VSMC) proteins mined from an analysis of the Human Protein Atlas. Two comparison were made: a) immunohistochemistry for 16 proteins in brain, heart, esophagus, bladder, stomach, lung, kidney, and aorta enabled comparison between human and mouse of protein localization in VSMC and non-vascular SMC; and b) multi-species primary protein sequence analysis of an expanded set vascular molecules enabled comparison between VSMC sequences among vertebrate species. In total, three dimensions of diversity were uncovered. First, a significant number of factors show human/mouse differences in cellular expression; these differences occurred in both VSMC and non-vascular SMC in an organ and cell-type dependent fashion. Many markers demonstrated notable cell-to-cell and regional heterogeneity in VSMC of the aorta and non-vascular SMC of the esophagus, bladder, and stomach. Second, species specificity can arise by genetic deletions as exemplified by the human protein adipogenesis regulatory factor (ADIRF), which is not present due to a large sequence gap in mice. Third, we describe significant cross-species protein sequence divergence in selected VSMC proteins which may result in altered orthologue function. In a sample of 346 vascular molecules, 15% demonstrate incomplete vertebrate species gene conservation. Divergence of predicted human/mouse VSMC protein sequences is higher than for endothelial proteins in all species examined. In the future, each of these three cross-species differences could be neutralized using gene manipulation, resulting in improved translational potential of murine experimental models.