The human FK506-binding proteins: characterization of human FKBP19 Article (Accepted Version) Rulten, Stuart L, Kinloch, Ross A, Tateossian, Hilda, Robinson, Colin, Gettins, Lucy and Kay, John E (2006) The human FK506-binding proteins: characterization of human FKBP19. Mammalian Genome, 17 (4). pp. 322-331. ISSN 0938-8990 This version is available from Sussex Research Online: http://sro.sussex.ac.uk/id/eprint/22944/ This document is made available in accordance with publisher policies and may differ from the published version or from the version of record. If you wish to cite this item you are advised to consult the publisher’s version. Please see the URL above for details on accessing the published version. Copyright and reuse: Sussex Research Online is a digital repository of the research output of the University. Copyright and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable, the material made available in SRO has been checked for eligibility before being made available. Copies of full text items generally can be reproduced, displayed or performed and given to third parties in any format or medium for personal research or study, educational, or not-for-profit purposes without prior permission or charge, provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. http://sro.sussex.ac.uk The Human FK506-Binding Proteins: Characterisation of Human FKBP19 Stuart L Rulten1*, Ross A Kinloch2, Hilda Tateossian3, Colin Robinson2, Lucy Gettins2 and John E Kay4. 1Trafford Centre for Graduate Medical Education and Research, University of Sussex, Falmer, Brighton, BN1 9QG, UK. 2Discovery Biology Dept. Pfizer Global Research and Development, Ramsgate Rd, Sandwich, Kent, CT13 9NJ, UK. 3MRC Mammalian Genetics Unit, Harwell, Oxfordshire OX11 0RD, UK 4Brighton and Sussex Medical School, Falmer, Brighton, BN1 9PX, UK * Address for correspondence: Dr. S. L. Rulten The Trafford Centre for Graduate Medical Education and Research University of Sussex Falmer Brighton, BN1 9RY Tel: +44-1273-872892 Fax: +44-1273-872941 E-mail: [email protected]. Abbreviations: FKBP: FK506-binding protein; EST: expressed sequence tag 1 Abstract Analysis of the human repertoire of the FK506-binding protein family of peptidyl- prolyl cis/trans isomerases (FKBPs) has identified an expansion of genes that code for human FKBPs in the secretory pathway. There are distinct differences in tissue distribution and expression levels of each variant. We describe here the characterisation of human FKBP19 (Entrez Gene ID: FKBP11), an FK506-binding protein predominantly expressed in vertebrate secretory tissues. The FKBP19 sequence comprises a cleavable N-terminal signal sequence followed by a putative peptidyl-prolyl cis/trans isomerase domain with homology to FKBP12. This domain binds FK506 weakly in vitro. FKBP19 mRNA is abundant in human pancreas and other secretory tissues and high levels of FKBP19 protein are detected in the acinar cells of mouse pancreas. Keywords FK506, petidyl-prolyl cis/trans isomerase, PPIase, pancreas 2 Introduction The synthesis, folding and assembly of proteins involves some of the most essential and conserved mechanisms of life (Pahl et al., 1997). In eukaryotic systems, several families of chaperones and foldases have evolved to catalyse these processes (Fedorov and Baldwin, 1997). The folding of proline-containing polypeptides is catalysed by peptidyl-prolyl cis/trans isomerases (PPIases: Fischer et al., 1984). The PPIase families are classified by sequence homology and pharmacologically by their ability to bind the immunosuppressant compounds cyclosporin, FK506 and rapamycin, and are otherwise known as immunophilins (reviewed in Galat and Riviere, 1998, Galat, 2003). The FK506-binding protein (FKBP) family share a high degree of sequence and structural homology and PPIase activity that is specifically inhibited by FK506 or rapamycin (Kay, 1996). Since the discovery of the first FKBP (Siekierka et al., 1989), several members of this family have been characterised in humans and other organisms (Patterson et al., 2002, Galat, 2003). Each member of the FKBP family contains one or more PPIase domain, which shows high sequence homology with the most abundant member, FKBP12. Analysis of the genomes of eukaryotic model organisms shows correlations between genome size and the number of genes coding for FKBPs, with 4 members present in Saccharomyces cerevisiae, 7 in Drosophila melanogaster, and 9 in Caenorhabditis elegans (Kay, 2000). In general, eukaryotic organisms appear to have one or two FKBPs for every 2,000 genes in their genome. If this pattern applied to the human genome, containing around 30,000 genes (Pennisi, 2003), there could be up to 30 3 FKBP sequences in the human genome. Although many cellular processes involving FKBPs may be conserved from lower organisms, and degeneracy involving cyclophilins or parvulins as alternative PPIases may significantly reduce this predicted number. This report describes a survey of the human genome for FK506-binding proteins and the biochemical characterisation of a novel member, FKBP19. Methods Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich Company Ltd, Poole, UK. Cloning IMAGE clones containing FKBP sequences were obtained from Research Genetics Inc. (Huntsville, AL, USA). These included IMAGE clone 269896 (accession numbers N24885 and N36303), containing the full-length FKBP19 coding sequence, clone 323084 (accession number W42674), containing a partial FKBP22 sequence, and clones 2518850 and 1857997 (accession numbers AI879695 and AI271550), which contained overlapping sequences covering the entire FKBP23 coding sequence. The FKBP23 clones shared a common internal StuI site. A BamHI-StuI fragment of the IMAGE clone 2518850 insert, and a StuI-NotI fragment of clone 1857997 were generated. A three-way ligation of these two fragments with a BamHI-NotI digested pET21a expression vector produced a full-length human FKBP23 clone. Northern Blotting Probes were generated to the open reading frames of each FKBP variant shown by PCR and radiolabelled with α-32P dCTP using a Megaprime labelling kit (Amersham Pharmacia Biotech Ltd. Little Chalfont, UK). These were then used to probe a Human 4 Multiple Tissue Northern and Human RNA Master Blot (Clontech) using ExpressHyb (Clontech), according to the recommended protocols. Washed blots were exposed to a phosphorimager screen (Molecular Dynamics, Sunnyvale, CA). In vitro transcription/translation of hFKBP19 A full-length FKBP19 expression construct was generated by PCR using primers FKBP19/3 and FKBP19/4 (see figure 3) and the resulting product subcloned into pET21a using restriction sites incorporated into the primer sequences. The construct was then used as a template in the TNT-T7 Quick Coupled Transcription/Translation System (Promega UK Ltd, Southampton, UK). Translation products were radiolabelled with 35S-methionine (Amersham Pharmacia Biotech Ltd. Little Chalfont, UK). Post-translational peptide cleavage was obtained by carrying out the coupled reaction in the presence of Canine Pancreatic Microsomal Membranes (Promega). Expression constructs Full-length expression constructs of FKBP19 and FKBP23 produced insoluble material in E.coli, so PCR primers (Genosys Biotechnologies Ltd, Cambridge, UK) were designed to amplify regions coding for the PPIase domain of human FKBP19 ( GAGGCTGGGCTCGAAACCG andGCCCTTCACCAGCTTTAGCC) and the “mature” FKBP23 (complete ORF minus the first 24 amino acids, using primers 5’- CCCATATGAGACAAAAGAAAGAGGAGAGC-3’ and 5’- CCCTCGAGTAGTTCATCGTGTTGGTATAC-3’). An in-frame start codon was incorporated, where necessary, into PCR primers, as part of an NdeI site, and an XhoI site was incorporated at the 3’ end, replacing the native stop codon. The resulting PCR fragment was cloned into pET21a (Novagen, Madison, WI, USA), such that the FKBP coding sequences were in-frame with a C-terminal His-tag sequence. A similar His- 5 Tagged expression construct was generated from the full-length coding region of human USA-CYP for use as a negative control for binding studies as previously described (Pemberton et al., 2003). An FKBP13 expression construct was made for use as a positive control by amplifying a human FKBP13 fragment coding for the “mature”, soluble form of the protein (i.e. the full-length minus the first 21 amino acids) with primers 5’-CCGAATTCACGGGGGCCGAGGGCAAAAGG-3’ and 5’- CCAAGCTTCAGCTCAGTTCGTCGCTCTAT-3’. The fragment was subcloned into pET21a using the EcoRI and HindIII sites incorporated into the primer sequences. Expression and purification of human PPIases BL21(DE3) cells (Novagen) transformed with each expression construct were grown in flasks, shaking at 200 rpm, at 37°C in 2YT broth supplemented with 50 µg/ml ampicillin, 1% glucose. Expression was induced at an OD600 of 0.8 with 1mM IPTG. Cells were harvested 2.5 hours after induction. Inclusion body preps were made from FKBP19-expressing E.coli as follows: 1g of cells were lysed by sonication in 10 ml 50 mM sodium phosphate (pH 7.0), 300 mM NaCl, 0.5 mM PMSF, and centrifuged at 1000g, 15 minutes, to pellet inclusion bodies. These were washed twice in lysis buffer and resuspended in lysis buffer containing 6 M guanidine-HCl. After 4 hours mixing at 4º, the samples were centrifuged again, and the supernatant