Monoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133)

Abstract

The pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.

Prominin‐1 (CD133) modulates the architecture and dynamics of microvilli

“…Given that MDCK cells express both canine prominin‐1 and prominin‐2 (K.T. and D.C., data not shown), we decided to study the consequences of the lack of human prominin‐1 in HSCs.…”

“…CHO cells were cultured in Ham's F‐12 nutrient mixture (+ l ‐glutamine, GIBCO) containing 10% FCS and 100 U/mL penicillin and 100 μg/mL streptomycin. Both cell lines were maintained in a humidified incubator at 37°C under 5% CO 2 atmosphere as described previously …”

Analysis of radiosensitivity of cancer stem‐like cells derived from canine cancer cell lines

“…Despite this genomic feature, the amino acid sequence is poorly conserved among CD133 gene products . The low level of amino acid conservation of CD133 between species may explain the lack of cross‐species immunoreactivity of specific CD133 antibodies between canine and mouse proteins . Further analysis is necessary to detect the protein expression of CD133 in CSCs.…”

“…37,38 The low level of amino acid conservation of CD133 between species may explain the lack of cross-species immunoreactivity of specific CD133 antibodies between canine and mouse proteins. 39 Further analysis is necessary to detect the protein expression of CD133 in CSCs. Moreover, other genes, such as Oct-4, Sox-2, Notch1, and c-myc, which are associated with the maintenance of stem cells.…”

The identification of a novel antibody for CD133 using human antibody phage display

“…The pentaspan transmembrane protein CD133 has been used extensively as a marker to identify and isolate prostate stem cells and cancer/stem initiating cells . Possessing an extracellular N‐terminal domain (EC1), two large heavily glycosylated extracellular loops (EC2 and EC3), and often existing as different splice variants, the biological function of CD133 is unknown . Identified originally as an antigen present on the surface of CD34 pos hematopoietic stem cells, the expression of CD133 has been documented in adult stems cells and cancer stem/initiating cells from a number of diverse tissue and cancer types .…”