by Gary Ciment, Ph.D., Scientific Director, Aves Labs, Inc.
Amyloid Precursor Protein (APP) is a 770-amino acid, single-pass transmembrane glycoprotein (Figure 1). Notoriously, APP can be proteolytically cleaved in brain tissue and elsewhere to give rise to two overlapping peptides implicated in the etiology of Alzheimer's disease (Nunan and Small, 2000; Selkoe, 2000). These two peptides are referred to as Ab1-40 and Ab1-42 (or -43), corresponding to residues 672-711 and 672-713 (or 714), respectively, and both straddle the extracellular-transmembrane border at G700.
Until recently, the major focus of APP-related research has been, understandably, on the Ab peptide domains. These peptides are cleaved from APP by a set of membrane-associated proteinases (i.e., secretases a and g) to give rise to the Ab peptides that spontaneously aggregate extracellularly. These aggregates form the distinctive amyloid plaques that are pathognomonic for Alzheimer's disease in human brain biopsy and autopsy slices (Selkoe, 2000; Hardy and Selkoe, 2002).
One enduring mystery of APP, therefore, concerns the function (or functions) of the rest of the protein. There are many reasons to believe, of course, that APP is around for more than just causing this devastating dementia. APP is widely expressed and highly conserved throughout the animal kingdom, from insects to humans (Coulson, et al., 2000). It is expressed in a wide variety of fetal tissues, with particularly high expression in brain, kidney, heart and spleen (Weidemann et al., 1989). There are sequences within the APP protein, moreover, that share homologies with known functional domains of other proteins. The extracellular E1 and E2 domains of APP, for example, show significant sequence homologies to functional domains within the ubiquitin gene product (Olsen and Lima, 2013). In this case, these regions are believed to be heparin- and metal-binding domains and are good candidates for mediating protein-protein interactions. A Kunitz (serine proteinase) inhibitor domain located between the E1 and E2 domains of APP provides another potential insight about APP functionality. This domain within APP, however, is unlikely to inhibit the a- and g-secretases known to generate the Ab peptides, since these are non-serine proteinases, and are not inhibited by the Kunitz serine proteinase inhibitor.
Various other studies suggest that non-Ab regions of APP may play a role in nervous system repair. It has been well established that traumatic brain injury causes the up-regulation of APP in humans and mice (Hortobagyi et al., 2007). Moreover, soluble extracellular regions of APP can act to ameliorate the effects of brain trauma (Thornton et al., 2006; Corrigan et al., 2012), suggesting some sort of protective effect. A number of studies indicate that the E1 and E2 heparin- and metal-binding domains of APP are involved in this protection, although the mechanism of this protection is not clear (c.f., Zhou et al., 2011).
On the other hand, the short intracellular domain of APP also seems to play a role in brain morphogenesis (Klevanski et al., 2015). Here, the very highly conserved YENPTY sequence on APP (which is contained within the peptide recognized by Aves' cAPP antibody) seems to be necessary for proper morphogenesis of neuromusclular junctions and the hippocampus. Knockout mice lacking the YENPTY motif demonstrate muscular weakness, as well as deficits in hippocampus-related behaviors, such as spatial learning.
Finally, in an intervertebrate model of neuronal migration, APP has been found to play a role in regulating the extent of neuroblast movement along the gut. This regulation seems to involve an intracellular domain within APP that interacts with the heterotrimeric G protein, Goa (Swanson et al., 2005; Ramaker et al., 2016; Ramaker and Copenhaver, 2017). In mammals, soluble extracellular fragments of APP bind with high specificity to another protein shown to be involved in neuronal migration, SorLA/LR11 (Andersen et al., 2006).
With the goal of developing antibody reagents against different functional regions of APP, we began by plugging the human APP sequence (NCBI accession # NP_000475.1) into our proprietary Immunogenicity Algorithm®. From the initial list of 30 candidates, about a dozen peptide sequences were chosen, making sure we covered the entire length of this gene product. For technical reasons, we added a cysteine and a synthetic spacer amino acid to the N-terminus of this sequence to improve conjugation and affinity purification efficiencies. KLH-conjugates of these peptides were then injected over a 7-week period into the breast muscles of laying hens, and eggs were collected after the 4th injection. Affinity purified antibodies were then prepared and sent to various collaborators to determine which antibodies demonstrated useful immunohistochemical signals. Once the useful antibodies were identified, we performed additional injections into the same hens used to prepare the initial antibody preparations, collected additional eggs, and performed additional affinity purified antibodies.
In sum, Aves Labs offers a variety of antibodies specific to various peptide sequences within the APP gene product, and these antibodies may be useful in the function or functions of this enigmatic protein, whose misprocessing likely leads to a devastating human disease.
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