In search of alternatively spliced alpha-Klotho Kl1 protein in mouse brain.

Alpha-Klotho is a multi-functional protein essential for maintenance of a myriad of cell functions. αKlotho is a single transmembrane protein with a large extracellular segment consisting of two domains (termed Kl1 and Kl2) which is shed into the extracellular fluid by proteolytic cleavage to furnish circulating soluble αKlotho. Based on cDNA sequence, an alternatively spliced mRNA is predicted to translate to a putative soluble αKlotho protein in mouse and human with only the Kl1 domain that represents a "spliced αKlotho Kl1" (spKl1) and is released from the cell without membrane targeting or cleavage. The existence of this protein remains in silico for two decades. We generated a novel antibody (anti-spE15) against the 15 amino acid epitope (E15; VSPLTKPSVGLLLPH) which is not present in Kl1 or full-length αKlotho and validated its specific reactivity against spKl1 in vitro. Using anti-spE15 and two well-established anti-αKlotho monoclonal antibodies, we performed immunoblots, immunoprecipitation, and immunohistochemistry to investigate for expression of spKl1 in the mouse brain. We found anti-spE15 labeling in mouse brain but were not able to see co-labelling of Kl1 and spE15 epitopes on the same protein, which is the pre-requisite for the existence of a spKl1 polypeptide, indicating that anti-spE15 likely binds to another protein other than the putative spKl1. In isolated choroid plexus from mouse brain, we found strong staining with anti-spE15, but did not find the spliced αKlotho transcript. We conclude that using reliable reagents and inclusion of proper controls, there is no evidence of the spKl1 protein in the mouse brain.