Distinct and Conserved Prominin-1/CD133–Positive Retinal Cell Populations Identified across Species

Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133) plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts—except for the axolotl—were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells), but they also marked distinct subdivisions of the inner nuclear layer (INL). In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b), a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6–positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1–positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1–positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1–expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies

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

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

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

<div><p>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.</p></div

Homemade mouse monoclonal antibody 80B258 and rabbit antiserum αhE2 fail to detect canine prominin-1.

<p>(<b>A, B</b>) MDCK cells stably transfected with human or mouse prominin-1, canine prominin-1-GFP as well as wild type cells (MDCK) were analyzed either by immunocytochemistry (A) or immunoblotting under reducing conditions (B) using mouse mAb 80B258 or rabbit antiserum αhE2. As a negative control, only the secondary antibody (as indicated) was used (A, B). For immunocytochemistry, cells were counterstained with DAPI (A). For immunoblotting, β-actin was used as loading control (black arrowhead). The arrow indicates the plasma membrane-associated form of prominin-1, and the open arrowhead indicates its endoplasmic reticulum-associated form. The original and uncropped blots are presented in in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164079#pone.0164079.s005" target="_blank">S5 Fig</a>. Scale bar, 30 μm.</p

Studies on canine tissues and/or cell lines using anti-prominin-1 antibodies.

<p>Studies on canine tissues and/or cell lines using anti-prominin-1 antibodies.</p

The commercial anti-human and anti-mouse antibodies fail to detect canine prominin-1 by immunoblotting.

<p>(<b>A-C</b>) Detergent lysates prepared from MDCK cells stably transfected with human or mouse prominin-1, canine prominin-1-GFP as well as wild type cells (MDCK) were analyzed by SDS-PAGE under reducing (A, C) and non-reducing (B) conditions and immunoblotting using mAbs AC133, AC141, 293C3, and 13A4 or polyclonal antibody against GFP. β-actin and α-tubulin were used as loading controls. As negative controls, only the secondary antibody (as indicated) was used (C). (<b>D</b>) Lysates from canine prominin-1-GFP transfected cells were incubated with (+) or without (–) PNGase F prior to immunoblotting with anti-GFP antibody or others (as indicated). The arrow and open arrowhead indicate the plasma membrane-associated form and endoplasmic reticulum-associated form of prominin-1, respectively. Asterisk indicates potential disulfide-bridged prominin-1 dimers or multimers, and the black arrowhead shows deglycosylated prominin-1. Molecular mass markers (kDa) are indicated. The original and uncropped blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164079#pone.0164079.s005" target="_blank">S5 Fig</a>.</p

Comparison of the amino acid sequence of canine, human and mouse prominin-1.

<p>The canine prominin-1.s23 sequence determined in this study (GenBank Accession No. KR758755; top) was aligned with human (AF027208; middle) and mouse prominin-1.s2 (NM_001163577; bottom). Black and grey backgrounds indicate identical and similar amino acid residues, respectively; dashed line, putative signal peptide; solid blue lines, predicted transmembrane segments; solid red line, exon 3; asterisk stretch, cysteine-rich region; red C, conserved cysteine residue in extracellular domains; #, potential N-glycosylation site in canine prominin-1; yellow box, conserved lysine potentially involved in the interaction with HDAC6; green box, conserved Src/Fyn phosphorylation (P) site; and arrowhead, exon boundaries.</p

Comparison of canine prominin-1 with its human and mouse orthologs.

<p>(<b>A</b>) Membrane topology. Canine prominin-1 contains an extracellular N-terminal domain (EC1), five transmembrane segments (1–5) separating two small intracellular (IC1 and IC2), two large extracellular (EC2 and EC3) loops, and an intracellular C-terminal domain (IC3). The latter harbors a phosphorylation (P) site at tyrosine residue 829 for Src and Fyn tyrosine kinases. The asterisk stretch indicates a cysteine-rich region at the transition of the first transmembrane segment and IC1 domain. The EC2 and EC3 domains contain nine potential N-glycosylation sites (forks). The positions of the alternative exons 3 (purple), 11a (green), 19 (pink) and 22 (brown) found in the extracellular domains are indicated. (<b>B</b>) Genomic organization. The organization of human (h) and mouse (m) <i>PROM1</i> genes is shown and compared to the canine equivalent (top panel). Vertical lines indicate exon boundaries and dark blue zones highlight the position of the transmembrane segments. Exon numbering begins with the one bearing the start codon. For simplicity, the details of the 5’-untranslated region (UTR) are not shown (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164079#pone.0164079.s001" target="_blank">S1B Fig</a>). Facultative exons included in the coding sequences are depicted in different colors and amino acid sequences of exons 3, 11a, 19 and 22 are indicated in parentheses (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164079#pone.0164079.s002" target="_blank">S2A Fig</a>). The new canine prominin-1 splice variants (s22, s23) and two additional predicted ones (<i>X1</i>, <i>X2</i>) including their GenBank accession numbers are presented (bottom panel). The presence (+) or absence (−) of an exon and the consequential protein sequence length (number of amino acids) are indicated. T represents the stop codon within exon 27. (<b>C</b>) Amino acid identity. The percentage of amino acid identity of each individual structural domain of canine (c) prominin-1 with its corresponding counterparts in human and mouse orthologs is presented.</p

Commercially available anti-human and anti-mouse antibodies fail to detect canine prominin-1 by cytochemistry and flow cytometry.

<p>(<b>A-C</b>) The MDCK cells stably transfected with human or mouse prominin-1, canine prominin-1-GFP as well as wild type cells (MDCK) were analyzed either by immunocytochemistry (A, B) or flow cytometry (C) using mAbs AC133, AC141, 293C3 and 13A4. For immunocytochemistry, cells were either cell surface labeled in the cold (A) or permeabilized with saponin after fixation (B). As negative controls, only the secondary antibody was used, and cells were counterstained with DAPI (A, B). Scale bars, 30 μm. For flow cytometry, cells were directly incubated with fluorochrome (PE or APC)-coupled mAbs as indicated. Cells expressing prominin-1-GFP were examined using the FITC channel. The vertical dashed lines indicate the cut-off for cells positive for a given prominin-1. These were established using unstained cells or those labeled with the irrelevant anti-human CD34 antibody conjugated to the appropriate fluorochrome (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164079#pone.0164079.s004" target="_blank">S4 Fig</a>). The color code indicates the corresponding cell line used, and the percentage of positive cells is specified in each panel.</p