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Biochimica et Biophysica Acta 1591 (2002) 175–179 www.bba-direct.com Short sequence-paper ABCG4: a novel human white family ABC-transporter expressed in the brain and eye

Susan Oldfield *, Christopher A. Lowry, Jon Ruddick, Stafford L. Lightman

University Research Centre for Neuroendocrinology, BRI, University of Bristol, Marlborough Street, Bristol BS2 8HW, UK Received 24 July 2001; received in revised form 11 April 2002; accepted 24 May 2002

Abstract

White family transporters are a group of ATP-binding cassette (ABC) that show to the Drosophila white product. The Drosophila is a subunit of heterodimeric transporters of precursors for eye-pigment synthesis. A novel, human member of this family (ABCG4) has been identified. Northern blotting shows that ABCG4 is expressed specifically in the brain and the eye. D 2002 Elsevier Science B.V. All rights reserved.

Keywords: White gene; ABC-transporter; ABCG1; Sterol

ATP-binding cassette (ABC) transporters form a large and its murine counterpart is known as ABCG1 or ABC8. superfamily of proteins that mediate the transport of a large Northern blotting has shown that the mRNAs encoding variety of diverse molecules across biological membranes in ABCG1 are present in several tissues, predominantly brain, an ATP-dependent manner [1]. The functional transporters spleen and lung [10]. contain two ATP-binding folds and two transmembrane The function of ABCG1 is unknown, although there is domains each with, usually, five or six membrane spanning evidence that suggests that it may have a role in cholesterol helices. The ATP-binding fold contains the conserved homeostasis. Transcription of ABCG1 is up-regulated in Walker A and Walker B nucleotide binding motifs [2] and lipid-loaded macrophages [12,13] and this has been shown an additional, highly conserved ‘ABC signature sequence’ to be dependent on the presence of nuclear oxysterol N-terminal to the Walker B motif is characteristic of this receptors (LXRs) [12]. Furthermore, using an antisense group of transporters. oligonucleotide, Klucken et al. [13] suppressed the expres- White family ABC-transporters show homology to the sion of ABCG1 and inhibited both cholesterol and phos- Drosophila white gene product [3,4]. These proteins are half pholipid transport. Recently, several splice variants of transporters in that they have six transmembrane domains ABCG1 mRNA have been described, which encode pro- and hence must dimerise to form functional, ATP-dependent teins that differ at their N-terminus [14,15]. transporters. The Drosophila white gene product heterodi- The mammalian dimerisation partners of ABCG1 are merises with the products of the related , scarlet [5] unknown. A cDNA encoding a second human white-related and brown [6], to give functional transporters that were protein, ABCG2 (BCRP, MXR1), which is involved in drug originally thought to transport the eye pigment precursors, resistance, has been cloned [16,17] and genomic sequencing tryptophan and guanine, respectively, into retinal cells [7,8]. suggests the existence of other members of the family. However, recent work by MacKenzie et al. [9] suggests that Indeed, since our identification of ABCG4 cDNA reported white may be involved in the transport of metabolites, such here, two further human white family cDNAs, ABCG5 and as 3-hydroxykynurenine, across intracellular membranes. ABCG8 [18,19] have been cloned. In order to clone novel Human and murine homologues of white have been cloned cDNAs of the white family, a database search was carried [10,11]. The human white gene has been named ABCG1 out to identify sequences with homology to ABCG1 with the hope of finding a related expressed sequence tag (EST) * Corresponding author. Tel.: +44-117-928-2181; fax: +44-117-928- that could be used to screen a cDNA library. This approach 2080. identified a partial cDNA clone (accession no. AL137563) E-mail address: [email protected] (S. Oldfield). that had been isolated from a human testis library [20]. The

0167-4889/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S0167-4889(02)00269-0 176 S. Oldfield et al. / Biochimica et Biophysica Acta 1591 (2002) 175–179 first 1300 nucleotides of this clone had 73% sequence sortium [21] and restriction mapping showed the insert size identity to the 3Vhalf of the coding region of ABCG1 but to be approximately 3.5 kb, long enough to be a full-length there was little similarity within the 3VUTRs. A subsequent cDNA encoding an ABCG1 homologue. The insert was database search was carried out to look for EST sequences fully sequenced on both strands and the sequence deposited with identity to the 3Vend of AL137563. This yielded a on the EMBL database with accession no. AJ300465. The sequence of 154 base pairs (accession no. AA912226), with sequence contained an open reading frame coding for a identity to the extreme 3Vend of AL137563. This sequence predicted protein of 627 amino acids in length. It has been was derived from a clone from a dorsal root ganglion named ABCG4 as a search of the database library. The clone was obtained from the I.M.A.G.E. con- maps this cDNA to a gene already designated ABCG4 on an

Fig. 1. Alignment of the amino acid sequence of ABCG4 with that of ABCG1. The first 19 amino acids of human ABCG4, shown in italics, give the sequence starting from the deduced translational start site. The sequence of ABCG1 is taken from Chen et al. [11] but with the N-terminus extended to take account of the 5VRACE results of Langmann et al. [23].( ) Walker A and B nucleotide binding motifs. ( ) ABC-signature sequence. ( ) Transmembrane helices. The underlined 12 amino acids of ABCG1 are included or excluded in alternative splice variants. S. Oldfield et al. / Biochimica et Biophysica Acta 1591 (2002) 175–179 177 incompletely sequenced genomic clone derived from chro- Following hybridisation, blots were washed in 0.1 Â SSC mosome 11. containing 0.1% SDS at 50jC. A mouse EST with high homology to the 5Vend of human On probing the human multi-tissue blot for ABCG4, a ABCG4 was also identified, obtained from I.M.A.G.E. and band of just under 4 kb was detected, exclusively in the lane fully sequenced (accession no. AJ426047). Alignment of the containing mRNA from brain (Fig. 2a). The size is consistent murine and human sequences indicated that the human with that expected for a mRNA from which a 3.5 kb cDNA sequence was probably missing the extreme 5Vend and that was cloned, allowing for the presence of a poly A tail. A this region included the true translational start site: in fact, the fainter, larger band was present in the thymus lane. Splice first base pairs of the human clone are the TG of the initiating variants of ABCG1 have been described [14] and this may be ATG. Furthermore, during revision of this paper, Engel et al. a splice variant or simply a nonspecific band. Subsequent [22] carried out 5VRACE using the sequence described here reprobing of the blot for actin mRNA indicated that similar for primer design and extended the sequence by 12 base pairs, amounts of RNA were present in each lane. As Drosophila giving the same translational start point as deduced above and white is important in Drosophila eye, it was of interest to resulting in an open reading frame encoding a polypeptide of look for expression of ABCG4 in the human eye. As shown 646 amino acids. ABCG4 is closely related to ABCG1 with 74% identity, 81% similarity at the amino acid level. ABCG1 and ABCG4 are much more closely related to each other than either is to any other member of the ABCG family. Fig. 1 shows the alignment of the deduced amino acid sequences of human and murine ABCG1 and 4. The human ABCG1 sequence is based on that of Chen et al. [11] but with the N-terminus extended to take into account the 5V RACE results of Langmann et al. [23], which indicate an open reading frame that commences MACLMAAF... The N-terminal sequen- ces of ABCG1 and ABCG4 are totally distinct and that of ABCG4 is shorter than that of ABCG1. Furthermore, the N- terminus of ABCG4 is unrelated to any of the N-termini of splice variants of ABCG1 [14,15]. The sequences are also divergent between amino acids 320 and 363 and between 571 and 605 of ABCG4. The former sequence is also the position of a splice variant in human ABCG1, which leads to the inclusion or exclusion of a dodecapeptide [11].Itis noteworthy that both ABCG1 and ABCG4 differ between species within these regions. Northern blot analysis of human and rat tissues was performed to determine in which tissues ABCG4 and ABCG1 are expressed. The 3V UTRs of ABCG1 and 4 have very low homology to each other and hence probes from these regions were used to allow specific detection of the two transporters independently. DNA fragments corre- sponding to nucleotides 2200–2931 of human ABCG4 and nucleotides 2316–3115 of mouse ABCG4 were amplified by PCR and used directly or cloned into pBluescript KSII+. For ABCG1, nucleotides 2082–2592 of human ABCG1 (accession no. X91249) and nucleotides 2082–2974 of rat ABCG1 (accession no. AJ303374) were amplified from human hypothalamic and rat brain cDNA, respectively, Fig. 2. Expression pattern of ABCG4. (a) A commercial, human multi- tissue Northern blot (Clontech) was probed with a cDNA fragment from the cloned into pBluescript KSII+ and sequenced to confirm 3VUTR of ABCG4 (top panel). For comparison, the blot was reprobed with their identities. Probes were radiolabelled using a Redi- a fragment of the 3VUTR of ABCG1 (middle panel) and with an actin prime kit (Amersham) and hybridised overnight to a com- cDNA as a loading control (bottom panel). A blot of human retinal fractions mercial multi-tissue, human poly A + Northern blot was also probed for ABCG4. (b) Northern blots of poly A + RNA from a (Clontech) or to blots of poly A + RNA prepared from variety of rat tissues were hybridised with probes specific for ABCG4 and ABCG1, respectively. Le, leucocytes; Lu, lung; P, placenta; SI, small various rat tissues and from human retina. RNAs were intestine; Li, liver; K, kidney; Sp, spleen; T, thymus; C, colon; SM, skeletal separated by electrophoresis through formaldehyde gels muscle; H, heart; B, brain; Te, testis; Th, thyroid; E, eye; CR, choroid and transferred to nylon membrane by capillary blotting. retina; NR, neural retina. 178 S. Oldfield et al. / Biochimica et Biophysica Acta 1591 (2002) 175–179 in Fig. 2a, ABCG4 mRNA is present in neural but not in white locus of Drosophila melanogaster, J. Mol. Biol. 180 (1984) choroid retina. The presence of ABCG4 in brain and neural 437–455. [4] M. Pepling, S.M. Mount, Sequence of a cDNA from the Drosophila retina but not in other tissues examined suggest that its melanogaster white gene, Nucleic Acids Res. 18 (1990) 1633. expression is confined to nervous tissue. Similar results were [5] R.G. Tearle, J.M. Belote, M. McKeown, B.S. Baker, A.J. Howells, + obtained on probing a Northern blot of poly A RNA from Cloning and characterization of the scarlet gene of Drosophila mela- various rat tissues for ABCG4 (Fig. 2b).Abandwas nogaster, Genetics 122 (1989) 595–606. detected in lanes containing brain or eye RNA but was not [6] T.D. Dreesen, D.H. Johnson, S. Henikoff, The brown protein of Dro- sophila melanogaster is similar to the white protein and to components present in any of the other lanes of heart, kidney, spleen, of active transport complexes, Mol. Cell. Biol. 8 (1988) 5206–5215. liver, lung or testis RNA. [7] G.D. Ewart, D. Cannell, G.B. Cox, A.J. Howells, Mutation analysis of This highly restricted tissue distribution is in contrast to the traffic ATPase (ABC) Transporters involved in uptake of eye pig- that of ABCG1 as shown when the human multi-tissue blot ment precursors in Drosophila melanogaster,J.Biol.Chem.269 was hybridised with a probe specific for ABCG1 (Fig. 2a). (1994) 10370–10377. [8] K. Summers, A. Howells, N. Pyliotis, Biology of eye pigmentation in This detected a band of approximately 3.5 kb in most tissues. insects, Adv. Insect Physiol. 16 (1982) 119–166. Highest levels were detected in the lung, placenta, liver, [9] S.M. Mackenzie, A.J. Howells, G.B. Cox, G.D. Ewart, Sub-cellular kidney and skeletal muscle but it was also present in the localisation of the white/scarlet ABC transporter to pigment granule spleen, thymus and, faintly, in heart and brain. In the rat, membranes within the compound eye of Drosophila melanogaster, ABCG1 bands were detected predominantly in the eye, liver, Genetica 108 (2000) 239–252. [10] J.M. Croop, G.E. Tiller, J.A. Fletcher, M.L. Lux, E. Raab, D. lung and spleen with weaker bands in the kidney and brain Goldenson, D. Son, S. Arciniegas, R.L. Wu, Isolation and character- (Fig. 2b). Other workers have also found ABCG1 mRNA in ization of a mammalian homologue of the Drosophila white gene, most tissues [10,11], although Croop et al. [10] found the Gene 185 (1997) 77–85. highest expression to be in the brain, spleen and lung. [11] H. Chen, C. Rossier, M.D. Lalioti, A. Lynn, A. Chakravarti, G. Perrin, However, their work was carried out before other members S.E. Antonarakis, Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to 21q22.3, Am. J. of the ABCG family were identified and the probes used may Hum. Genet. 59 (1996) 66–75. not have been specific for ABCG1. 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