Broad Expression Analysis of Human ANTXR1/TEM8 Transcripts Reveals Differential Expression and Novel Splizce Variants

Micaela Vargas,Stephen H Leppla,G Jilani Chaudry,Raghavendra Karamsetty,Wanjin Hong

doi:10.1371/journal.pone.0043174

Abstract

Tumor endothelial marker 8 (TEM8; ANTXR1) is one of two anthrax toxin receptors; the other is capillary morphogenesis gene 2 protein (CMG2; ANTXR2). TEM8 shows enhanced expression in certain tumor endothelia, and is thought to be a player in tumor vasculature formation. However, a comprehensive expression profile of individual TEM8 variants in normal or cancerous tissues is lacking. In this work we carried out an extensive analysis of all splice variants of human TEM8 in 12 digestive tissues, and 8 each fetal and adult tissues, 6 of them cognate pairs. Using variant-specific primers, we first ascertained the status of full-length transcripts by nested PCR. We then carried out quantitative analysis of each transcript by real-time PCR. Three splice variants of TEM8 were reported before, two single-pass integral membrane forms (V1 and V2) and one secreted (V3). Our analysis has revealed two new variants, one encoding a membrane-bound form of the receptor and the other secreted, which we have designated V4 and V5, respectively. All tissues had V1, V2, V3, and V4, but only prostate had V5. Real-time PCR revealed that all variants are present at different levels in various tissues. V3 appeared the most abundant of all. To ascertain its functionality for anthrax toxin, we expressed the newly identified form V4 in a receptor-negative host cell, and included V1 and V2 for comparison. Cytotoxicity, toxin binding, and internalization assays showed V4 to be as efficient a receptor as V1 and V2.

Highlights

Tumor endothelial marker 8 (TEM8) was originally discovered by serial analysis of gene expression (SAGE) in endothelial cells of colon carcinomas [1]
The analysis revealed markedly elevated levels of TEM8 mRNA in these cells, and normal endothelial cells appeared to have little of it
Later findings showed that TEM8 mRNA shows elevated expression in certain other tumor endothelia [29]

Summary

Introduction

Tumor endothelial marker 8 (TEM8) was originally discovered by serial analysis of gene expression (SAGE) in endothelial cells of colon carcinomas [1]. The analysis revealed markedly elevated levels of TEM8 mRNA in these cells, and normal endothelial cells appeared to have little of it. These studies identified three splice variants of the gene, V1, V2, and V3. The variants encode proteins of 564, 368, and 333 residues, respectively. V1 and V2 are type-1 integral membrane proteins with a single tramsmembrane helix. V2 is identical to V1 up to residue 364, but the last four residues of V2 are unique, a consequence of differential splicing. V3 has a portion that is identical to the extracellular portions of V1 and V2, but its 13-residue carboxyl terminal segment is unique

Methods

Results

Conclusion