Identification and differential expression of three novel alternatively spliced isoforms of hematopoietic stem cell marker AC133
Jun, Lin; Shmelkov, Sergey V.; St Clair, Ryan; Shido, Koji; Walsh, Kathryn A.; Lam, George; Moussazadeh, Nelson; Gordon, Rachael; Rafii, Shahin
Blood 102(11): 330a
AC133 antigen is a 120kDa five transmembrane domain glycoprotein which is expressed on primitive cell populations, such as CD34 bright hematopoietic stem and progenitor cells, neural and endothelial stem cells, and other undifferentiated cells such as retinoblastoma and developing epithelium. AC133 antigen localizes to microvilli and other plasma membrane protrusions of stem/progenitor cells, and in the apical, but not the baso-lateral, membrane surface of some epithelial cells. AC133 antibody is used for positive selection of hematopoietic stem cells as an alternative to the widely used CD34. However, it is known that certain populations of cells which are positive for AC133 by RT-PCR, cannot be recognized by AC133 antibody. One explanation is that the antibody recognition sites are glycosilation-dependent epitopes, since AC133 sequence contains 8 N-linked glycosylation sites: all within the putative extracellular domains. Another possibility is that AC133 antibody may recognize only one of a few possible alternatively spliced forms. Yu et al. (2002) reported an alternative splicing form (AC133-2) that lacks exon 4, and concluded that AC133-2 might be the stem cell specific form. Here, we found three additional alteratively spliced isoforms of AC133, all differing in the C-terminal cytoplasmic tail. The previously reported AC133 contains a 50aa C-terminal cytoplasmic tail with five tyrosine residues (possibly indicating that the protein can transduce extracellular signals). Our novel isoforms have three tyrosine residues, indicating a high possibility of cellular signal transduction. Mutation analysis of these three tyrosine sites might help us to determine the function of AC133. RT-PCR analysis reveals a tissue specific expression of AC133 isoforms, suggesting that the protein might be an organ specific stem cell marker. Overexpression of AC133 isoforms fused with EGFP shows that they are all located to the cell membrane. It is still not clear which isoform is associated with hematopoietic stem cells. Overexpression combined with flow cytometry using AC133 antibody might allow us to determine hematopoietic stem cell specific isoform of AC133.