Conservation of a platelet activating domain of Aggrus/podoplanin as a platelet aggregation-inducing factor Mika Kato Kaneko a,b, ⁎ , Yukinari Kato a,b , Takashi Kitano a , Motoki Osawa a,c a Department of Experimental and Forensic Pathology, Yamagata University School of Medicine, 2-2-2, Iida-nishi, Yamagata 990-9585, Japan b Glycogene Function Team of Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), OSL C2, 1-1-1, Umezono, Tsukuba 305-8568, Japan c Department of Forensic Medicine, Tokai University School of Medicine, Boseidai, Isehara, Kanagawa 259-1193, Japan Received 7 January 2006; received in revised form 22 April 2006; accepted 25 April 2006 Available online 11 May 2006 Received by N. Saitou Abstract Human Aggrus/podoplanin is an identified platelet aggregation-inducing factor of cancer cells, which is also known as a specific marker of lymphatic endothelium. Human Aggrus was known to be expressed in seminoma, squamous cell carcinoma, malignant mesothelioma, sarcomas and several brain tumors. In our previous studies, the sialylated O-glycan of human and mouse Aggrus were shown to be critical for its platelet aggregation-inducing activity in the experiments using the glycosylation-deficient Chinese hamster ovary (CHO) cell lines. We newly cloned Aggrus homologues from rat, hamster, dog and bovine cDNAs, in addition to the human and mouse cDNAs, and confirmed there are three tandem repeats of the platelet aggregation-stimulating (PLAG) domain in Aggrus, which were conserved in all homologues. We found that bovine Aggrus has a sporadic deletion mutation in the first PLAG domain, and lacks platelet aggregation-inducing activity. We introduced point mutation in the PLAG domain of Aggrus and showed that either the first or last PLAG domain is critical for activity, but not the middle domain. In addition, we studied the molecular evolutionary process of the PLAG domain of Aggrus. The PLAG domain and its activity appeared after the divergence of avians and mammals. In conclusion, we provide evidence that Aggrus homologues conserved the segment of EDxxVTPG in their extracellular domain which are critical for their platelet aggregation-inducing activities. © 2006 Elsevier B.V. All rights reserved. Keywords: Platelet aggregation; PLAG domain; Homologous gene 1. Introduction Platelet aggregation is known to be related to cancer metastasis. Our previous studies have clarified that membranous 44-kDa and 36-kDa sialoglycoproteins, respectively of cancer cells of mice and humans, aggregated platelets with no relation to plasma compo- nents (Kato et al., 2003; Kaneko et al., 2004). Ectopic expression of these molecules, designated as Aggrus, was observed in various cancer cells, including testicular tumors, squamous cell carcino- mas, mesothelioma, and brain tumors, in which up-regulated ex- pression was evident (Kato et al., 2003, 2004, 2005; Kimura and Kimura, 2005; Martin-Villar et al., 2005; Mishima et al., 2006a,b; Wicki et al., 2006). Unique characteristics of Chinese hamster ovary (CHO) mutant cell lines Lec1, Lec2 and Lec8 revealed that sialylated O-glycan is critical for platelet aggregation-inducing ac- tivity (Kaneko et al., 2004). Homologous molecules to Aggrus have been identified inde- pendently in several mammals: T1α (Dobbs et al., 1988; Rishi et al., 1995), podoplanin (Breiteneder-Geleff et al., 1997) and PA2.26 (Gandarillas et al., 1997) are isolated from rat alveolar type- I cells in lung, rat glomerular epithelial cells (podocytes) or mouse keratinocytes. Other homologues include the canine receptor for the influenza C virus gp40 (Gandarillas et al., 1997) and its human homologue gp36 (Zimmer et al., 1999) and RANDAM-2 (Kotani et al., 2003), the membrane glycoprotein expressed in neuronal Gene 378 (2006) 52 – 57 www.elsevier.com/locate/gene Abbreviations: CHO, Chinese hamster ovary; PLAG, platelet aggregation- stimulating; PCR, polymerase chain reaction; RT, reverse transcriptase; ORF, open reading frame; PRP, platelet-rich plasma; PBS, phosphate buffered saline; SDS, sodium dodecyl sulfate; ECL, enhanced chemiluminescence kit; ERM, ezrin, radixin, moesin; WT, wild type; EST, expressed sequence tag; HMM, Hidden Markov Model; PAGE, polyacrylamide gel electrophoresis. ⁎ Corresponding author. Glycogene Function Team of Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technol- ogy (AIST), OSL C2, 1-1-1, Umezono, Tsukuba 305-8568, Japan. Tel.: +81 29 861 3197; fax: +81 29 861 3191. E-mail addresses: mk-kaneko@aist.go.jp, mikanekok@hotmail.com (M.K. Kaneko). 0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2006.04.023