Bovine intelectins: cDNA sequencing and expression in the bovine intestine q Stacey C. Blease a , Anne T. French a , Pamela A. Knight a , David L. Gally b , Alan D. Pemberton a, * a Division of Veterinary Clinical Sciences, University of Edinburgh, Royal (Dick) School of Veterinary Studies, Easter Bush Veterinary Centre, Roslin, Midlothian EH25 9RG, UK b Centre for Infectious Diseases, Royal (Dick) School of Veterinary Studies, Chancellor’s Building, University of Edinburgh, Edinburgh EH16 4SB, UK article info Article history: Accepted 15 June 2008 Keywords: Cattle Intelectin Lactoferrin Neutrophil elastase Intestine EHEC abstract Intelectins (Itlns) are lectins with potential roles in innate immunity, capable of binding bacteria via galactofuranose residues. Itlns also function as intestinal receptors for the antimicrobial glycoprotein lac- toferrin (Lf). Since Lf binds strongly to enterohemorrhagic Escherichia coli O157:H7 (EHEC), we aimed to determine the expression of Lf receptor in terminal rectum, the site of predilection of EHEC in cattle. We sequenced two bovine intelectins (Itln1 and Itln2) and showed that both were expressed in abomasum and rectum, but expression appeared minimal in the jejunum. There was significantly higher expression of Itln2 in terminal rather than proximal rectum. Lactoferrin was expressed in all samples examined. Thus, we have demonstrated two novel bovine Itlns and shown that they are expressed along with Lf in the gastrointestinal tract, where they may interact with microbial pathogens. Ó 2008 Elsevier Ltd. All rights reserved. Lactoferrin (Lf) is a potent antimicrobial glycoprotein, with mul- tiple functions. These include iron sequestration, LPS binding and impairment of the type III secretory apparatus of bacteria (Ellison, 1994; Ochoa et al., 2003). As well as being present in milk, Lf is se- creted by submucosal glands of the airway and intestine and is also stored in secretory granules of neutrophils (Ward et al., 2002). Lf present in bovine intestine has been shown to be located primarily on the epithelial surface overlying lymphoid follicles, e.g. Peyer’s patches in the ileum (Talukder et al., 2003), presumably bound by the intestinal Lf receptor, intelectin (Suzuki et al., 2005). Intelectin (Itln) has been characterised in a number of mamma- lian species, including man (Tsuji et al., 2001), mouse (Komiya et al., 1998), pig (Liao et al., 2007) and sheep (French et al., 2007), but not previously in cattle. It is expressed by cell types within the intestinal epithelium, including Paneth cells (Komiya et al., 1998) and goblet cells (Pemberton et al., 2004). Itln has also been found to localise within the epithelial brush border (Wrackmeyer et al., 2006), where it can interact with Lf and potentially other ligands. In particular, Itln has been shown to have a high affinity for galactofuranose (Galf) residues, which are present in the surface glycoproteins of numerous microorganisms (Tsuji et al., 2001). Mammals, however, lack the enzyme UPG-galactopyranose mutase (glf) which catalyses the conversion of galactopyranose to Galf. Thus, human Itln was shown (Tsuji et al., 2001) to bind cell wall proteins of Nocardia, and so Itln located within the brush border may potentially bind to bacteria either directly via Galf residues, or indirectly via Lf. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a dan- gerous environmental pathogen, for which cattle represent an important natural reservoir (Naylor et al., 2005). It has been shown that the bacterium preferentially colonises lymphoid follicles in the terminal rectum (Naylor et al., 2003). Moreover, it is known that Lf binds strongly to EHEC (Shin et al., 1998), and therefore it is possible that Lf bound to the epithelial surface promotes coloni- sation of the terminal rectum by EHEC via Lf receptors at the epi- thelial surface. Consequently, we aimed to determine whether the intestinal Lf receptor (i.e. Itln) was present at the terminal rectum. In this short communication, we describe the sequencing of two novel bovine intestinal intelectins and investigate their expression in rectal tissues, in comparison with that of Lf. In order to determine bovine Itln sequence(s), we utilised pub- licly available bovine EST data and the then current assembly of the bovine genome (Btau 2.0, March 2005; http://www.ensem- bl.org), in addition to sequences from our ongoing sequencing of sheep intelectins. This information was used to design primers to amplify partial or full coding regions. Primers were also designed for Lf, neutrophil elastase 2 and ATPase, with the aid of the freely available Primer 3 program (http://frodo.wi.mit.edu/cgi-bin/pri- mer3/primer3_www.cgi). Samples of bovine abomasum infected with Ostertagia ostertagi were donated by Dr. David Smith, More- dun Research Institute, for use as starting material, since nematode infection is known to increase levels of intelectin transcription (Pemberton et al., 2004). 0034-5288/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2008.06.002 q EMBL Accession numbers for bovine Itln1 and Itln2 are AM749042 and AM749043, respectively. * Corresponding author. Tel.: +44 131 650 7348; fax: +44 131 6501 3903. E-mail address: alan.pemberton@ed.ac.uk (A.D. Pemberton). Research in Veterinary Science 86 (2009) 254–256 Contents lists available at ScienceDirect Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc