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ated with antigen processing (TAP) which is a heterodi- mer comprised of TAP1 and TAP2 proteins. TAP is a member of the ATP-binding cassette (ABC) family of.
Immunogenetics (2002) 54:30–38 DOI 10.1007/s00251-001-0416-6

O R I G I N A L PA P E R

Aruna P.N. Ambagala · Zhengyu Feng Raul G. Barletta · Subramaniam Srikumaran

Molecular cloning, sequencing, and characterization of bovine transporter associated with antigen processing 2 (BoTAP2 ) Received: 30 August 2001 / Revised: 14 November 2001 / Published online: 2 March 2002 © Springer-Verlag 2002

Abstract The transporter associated with antigen processing (TAP) 1/2 heterodimer is essential for the transport of antigenic peptides from the cytosol into the lumen of the endoplasmic reticulum. Bovine herpesvirus 1 (BHV-1) inhibits bovine TAP (BoTAP) activity, as a means of down-regulating MHC class I expression on the cell surface, and hence evasion of the cytotoxic Tlymphocyte response of the host. Identification of BHV1 protein(s) responsible for TAP inhibition, and elucidation of the mechanism of TAP inhibition necessitate cloning and high-level expression of BoTAP1 and 2. In this study, we cloned and sequenced BoTAP2. Cytoplasmic RNA isolated from bovine peripheral blood mononuclear cells was used for cDNA synthesis. Rapid amplification of cDNA ends was used to amplify the 5′ and the 3′ ends of BoTAP2 cDNA. Based on the 5′ and 3′ sequences, primers were designed and the full-length BoTAP2 cDNA was PCR-amplified and sequenced. The full-length cDNA encodes a 719-amino acid polypeptide with a predicted molecular weight of Mr 79,000. BoTAP2 has ~80% homology, at the amino acid level, to its mammalian counterparts. Similar to human TAP2, BoTAP2 consists of seven putative transmembrane segments followed by an ATP-binding cassette. As expected, the level of BoTAP2 mRNA expression was up-regulated by treatment with recombinant bovine interferon-γ. In Northern blot analysis, BoTAP2 transcripts were detected in several bovine tissues with the highest level observed in jejunum. BoTAP2, when expressed as a green fluorescent fusion protein, exhibited a typical endoplasmic reticulum localization pattern. Keywords Bovine · TAP2 · Cloning · Sequencing · Expression A.P.N. Ambagala · Z. Feng · R.G. Barletta · S. Srikumaran (✉) Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Fair Street and East Campus Loop, Lincoln, NE 68583-0905, USA e-mail: [email protected] Tel.: +1-402-4723319, Fax: +1-402-4729690

Introduction The majority of cytotoxic T-lymphocytes (CTLs) recognize antigenic peptides in the context of major histocompatibility complex (MHC) class I molecules (Townsend et al. 1986; Zinkernagel and Doherty 1975). Felicitous cell surface expression of MHC class I molecules requires efficient assembly of class I-peptide complexes in the endoplasmic reticulum (ER). The peptides bound to MHC class I molecules are mainly generated in the cytosol by the proteasome (Heemels and Ploegh 1995; Momburg and Hammerling 1998). These peptides are translocated into the lumen of the ER by the transporter associated with antigen processing (TAP) which is a heterodimer comprised of TAP1 and TAP2 proteins. TAP is a member of the ATP-binding cassette (ABC) family of transporters (Androlewicz et al. 1993; Neefjes et al. 1993; Shepherd et al. 1993). ABC transporters, also known as traffic ATPases, comprise a large family of membrane-associated export and import systems present in prokaryotes and eukaryotes (Higgins 1992). As the name implies, they utilize the energy of ATP hydrolysis to pump substrate across the membrane against a concentration gradient. Each ABC protein is specific for a substrate or a group of related substrates that can range from inorganic ions to sugars, amino acids, complex polysaccharides, and peptides. ABC transporters contain four structural domains: two related and highly conserved nucleotide-binding domains (NBDs) and two hydrophobic transmembrane domains (TMDs). Each NBD consists of Walker A and Walker B motifs, two highly conserved ATP-binding sequences and the ABC transporter “signature” (Higgins et al. 1985; Hyde et al.1990; Walker et al. 1982). Most of the eukaryotic ABC transporters, such as the human mutidrug resistance P-glycoproteins and cystic fibrosis transmembrane conductance regulators (CFTRs), have all four domains fused into a single polypeptide. In contrast, some of the ABC transporters such as peroxisomal membrane proteins (PMPs), TAP, and TAP-like (TAPL) proteins consist of two separate subunits each containing one hydrophobic TMD and

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one NBD. Therefore PMPs, TAP, and TAPL are also called half-transporters. TAPL is a recently discovered ER resident protein which has high homology to TAP proteins (Kobayashi et al. 2000; Yamaguchi et al. 1999). However, unlike TAP1 and TAP2, human TAPL is located on Chromosome (Chr) 12 and not responsive to interferon (INF)-γ treatment. Therefore, TAPL is unlikely to play a role in the antigen presentation pathway. Proteins involved in antigen presentation are encoded by genes located in the MHC region of Chr 6 and 17 in humans and mice, respectively. Almost all the proteins involved in the MHC class I antigen presentation pathway are encoded by genes located in the MHC class I region, except low molecular-mass protein 2 (LMP2), LMP7, TAP1, and TAP2 which are encoded by genes located in the MHC class II region. The MHC in cattle is termed the bovine lymphocyte antigen system (BoLA) (Lewin et al. 1999). Unlike humans and mice, BoLA class II has two subdivisions: BoLA IIa and BoLA IIb. Bovine TAP2 (BoTAP2), LMP2, and LMP7 genes have been mapped to the BoLA IIb region which has segregated from the rest of the BoLA genes due to a large inversion in Chr 23 (Hess et al. 1999). Recent studies in our laboratory have revealed that bovine herpesvirus-1 (BHV-1) interferes with transport of peptides by BoTAP molecules as a means of evading the CTL response (Hinkley et al. 1998). Elucidation of the mechanism(s) of TAP inhibition and identification of BHV-1 protein(s) involved necessitates cloning and high-level expression of BoTAP1 and BoTAP2 molecules. In this study, we report cloning, sequencing, and characterization of BoTAP2.

Materials and methods Cells and tissues Bovine peripheral blood mononuclear cells (PBMCs) were isolated from blood collected from healthy cattle, by centrifugation on a Ficoll-Paque gradient (Amersham Pharmacia Biotech, Arlington Heights, Ill.). The cells were washed twice with ice-cold sterile phosphate-buffered saline (PBS; 130 mM NaCl, 1.55 mM KH2PO4, 5.1 mM Na2HPO4, pH 7) and cytoplasmic RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, Calif.) according to the manufacturer's instructions. Madin Darby bovine kidney (MDBK) and African green monkey kidney (COS-7) cells (both from ATCC, Manassas, Va.) were propagated as described previously (Nataraj et al. 1997). Recombinant bovine IFN-γ was a kind gift from Dr. L.A. Babiuk, VIDO, Saskatoon, Canada. MDBK cells were either untreated or treated with recombinant bovine IFN-γ, for indicated time periods. Different bovine tissue samples were collected at slaughter from a 10 month-old healthy calf and flashfrozen in liquid nitrogen. Total cellular RNA was purified from these tissues using TRizol reagent (Life Technologies, Grand Island, N.Y.).

Table 1 The sequences of primers designed for PCR amplification of BoTAP2 Primer

Sequence

FP1 FP2 FP3 RP1 RP2 RP3 RP4 RP5 RP6

5′ GGCTGAGCTCGGATACCA 3′ 5′ CTGATGAGTAACTGGCTTCC 3′ 5′ CACTGATCTCGCCATGCG 3′ 5′ TGATGGCGGGCACTGTAC 3′ 5′ ATCTCCCAAAGGACTGCCTG 3′ 5′ ACCACCTGACCAGCTTTCG 3′ 5′ CTTCGGCTCCAAAACTGC 3′ 5′ CTAAGGTTCCTGGATGCAGAC 3′ 5′ GGTGGAGGCAGATCTGGCTTTC 3′

forward gene-specific primers, FP1 and FP2 (Table 1), synthesized based on the 349-bp partial BoTAP2 cDNA sequence available at GenBank (accession number AF110317). Briefly, first-strand cDNA synthesis was performed using reverse transcriptase (RT) and oligo-dT adaptor primer (3′AP). The original mRNA template was then destroyed with RNase H. Specific cDNA was then amplified using FP1 and 3′ universal amplification primer (3′UAP) and subjected to electrophoretic analysis. To provide added specificity, a small agarose plug (5 µl) was removed from the band of interest and subjected to another round of PCR using FP2 and the 3′UAP. To ensure that the final 3′RACE product was in fact the 3′ end of BoTAP2, the 3′RACE products (obtained using FP2 and 3′UAP) were subjected to Southern blot analysis. Briefly, the 3′RACE products were size-fractionated by electrophoresis, blotted onto a nylon membrane (Micron Separation, Westborough, Mass.) and hybridized overnight with a 190-bp BoTAP2 probe. The BoTAP2 probe was generated by PCR amplification of MDBK genomic DNA using FP1 and RP1 primers. RP1 was also synthesized based on the partial BoTAP2 cDNA sequence available in GenBank (accession number AF110317). MDBK genomic DNA was isolated using TRizol reagent according to the manufacturer's instructions. Labeling the probe, hybridization, and washing were carried out as described under Northern blot analysis. The verified 3′RACE was cloned into pCR2.1 (Invitrogen, Carlsbad, Calif.) and sequenced. The sequence data were analyzed using the NCBI BLASTX program (http://www.ncbi.nlm.nih.gov/BLAST/) to confirm that the sequence had a considerable homology to known mammalian TAP2 sequences. Based on the sequence data, three reverse primers (RP2, RP3, and RP4; Table 1) were designed and used in 5′RACE. Briefly, cytoplasmic RNA was reverse-transcribed using RP4. After first-strand synthesis, the original mRNA was removed by treatment with RNase H and RNase T1. Excess dNTPs, enzymes, and RP4 were removed using the GLASSMAX DNA Isolation Spin Cartridge procedure. A homopolymeric tail was added to the 3′ end of the first-strand cDNA using TdT and dCTP. The amplification of the 5′ end was accomplished by subjecting the first-strand cDNA to nested PCR using 5′AP, 5′UAP, RP3, RP2, and RP1 primers. The amplified cDNA fragment was cloned into pCR 2.1 and sequenced. Based on 5′ and 3′ sequencing data, FP3 and RP5 primers (Table 1) were designed to include the full-length BoTAP2 gene. Full-length BoTAP2 was amplified using the Expand High Fidelity PCR system (Roche, Indianapolis, Ind.), cloned into pCR 2.1 and both positive and negative strands were sequenced. The primers were synthesized at IDT (Coralville, Iowa). The sequencing analyses were performed at the DNA Sequencing and Synthesis Facility at the Iowa State University, Ames, Iowa. The nucleotide sequence of BoTAP2 has been submitted to GenBank (accession number AF318024).

BoTAP2 cDNA cloning Cytoplasmic RNA isolated from bovine PBMCs was subjected to cDNA synthesis followed by rapid amplification of cDNA ends (RACE) using a 3′RACE system and 5′RACE system (Version 2) from Life Technologies. Taq DNA polymerase from Qiagen, was used in both 3′ and 5′RACE. 3′RACE was carried out using two

Construction of a plasmid encoding BoTAP2-EGFP fusion protein To clone the full-length BoTAP2 into pEGFP-N1 (Clontech, Palo Alto, Calif.), a forward primer (5′ CCCAAGCTTATGCGGAGCCCGGACCTG 3′) with HindIII site (underlined) and reverse

32 primer (5′ GGGGTACCGTCTCCGGGTCGCTGAG 3′) with KpnI site (underlined) were designed. Using the above primers and the Expand High Fidelity PCR system (Roche), the full-length BoTAP2 sequence was amplified and cloned into HindIII and KpnI sites of pEGFP-N1. The construct, pBoTAP2/EGFP, was subsequently sequenced to verify the integrity of the fusion protein.

croscope (Bio-Rad, Herculus, Calif.). Optical images were collected using a dual excitation/emission (450/490 nm for EGFP, and 640/690 nm for Cy5) by simultaneous display mode of the BioRad LaserSharp Program. The images were analyzed by Confocal Assistant 4.02, created and copyrighted by Todd Clark Brelje. Biocomputing

Northern blot analysis Cytoplasmic RNA isolated from MDBK cells (either untreated or treated with recombinant bovine IFN-γ, 4 µg per 5×106 cells in 5 ml of growth medium) or total cellular RNA from bovine tissues (15 µg/lane) was electrophoresed on a 1% agarose gel containing 6.7% formaldehyde and blotted onto a nylon membrane (Micron Separation) by standard upward capillary transfer in 20×sodium chloride sodium citrate (SSC) transfer buffer overnight. The membranes were baked for 2 h at 80°C and hybridized overnight in Rapid hyb buffer (Amersham Pharmacia Biotech). A BoTAP2 probe (~1.5 kb) was generated by performing PCR on full-length BoTAP2 using forward primer FP3 and reverse primer RP6. The FP3 and RP6 primers were chosen specifically not to amplify any sequence from the putative NBD. A 0.75-kb 18S rRNA probe was obtained by digesting the plasmid pN29III (kindly provided by Dr. R. Donis, University of Nebraska-Lincoln, Lincoln, Neb.) with BamHI and SphI (both from NEB, Beverly, Mass.). Random primer labeling of all the probes with Redivue α[32P]-dCTP was performed using the “rediprime II” DNA labeling system (both from Amersham Pharmacia Biotech) according to the manufacturer's instructions. The high-stringency washing conditions recommended for the Rapid hyb buffer were followed. For normalization of RNA amounts, BoTAP2 probe was stripped by pouring 500 ml of boiling 0.1% (w/v) SDS solution over the blot, washed with 2×SSC, and reprobed with 18S rRNA probe. Quantification of the 18S and BoTAP2 bands was performed using PhosphorImager SF (Molecular Dynamics, Sunnyvale, Calif.) Transient BoTAP2-EGFP expression and immunofluorescence For transient transfection and immunofluorescence, COS-7 cells were selected over MDBK cells for two reasons: (1) poor transfection efficiency of MDBK cells and (2) lack of availability of antibovine calreticulin or calnexin antibodies. COS-7 cells were transiently transfected with the pBoTAP2/EGFP construct using Superfect transfection reagent (Qiagen) according to the manufacturer's instructions. Briefly, COS-7 cells were seeded into 12-well plates containing sterile glass coverslips (2.5×104 cells/well) the day prior to transfection. One microgram of pBoTAP2/EGFP or pEGFP-N1 and 10 µl of Superfect reagent were mixed, incubated for 10 min at room temperature and diluted with 300 µl of serumcontaining growth medium. The cells were washed once with serum-free medium and DNA/Superfect complexes were added. After 3 h incubation at 37°C, the medium was removed and 1 ml of fresh growth medium was added. The cells were evaluated for green fluorescence under a LEICA DM IRB inverted fluorescence microscope (Leica, Deerfield, Ill.) at 12, 24, 36, and 48 h posttransfection. For immunofluorescence, the cells were rinsed with PBS, and fixed in 4% paraformaldehyde in Tris-buffered saline (TBS; 25 mM Tris, 150 mM NaCl, pH 7.4) and permeabilized with 100% methanol at –20°C. The samples were blocked overnight with 3% bovine serum albumin /0.05% Tween 20 in TBS. Blocking buffer was removed and the coverslips were incubated with goat anti-human calreticulin antibodies (Santa Cruz Biotech, Santa Cruz, Calif.) for 1 h. The coverslips were then washed three times in TBS and incubated in the dark with donkey anti-goat IgG-Cy5 antibodies (Jackson Laboratories, West Grove, Pa.) for 1 h. Following three extensive washes (twice in TBS and the last wash in PBS), the coverslips were mounted using Gel/Mount aqueous mounting medium (Biomeda, Foster City, Calif.). The samples were analyzed using a BioRad MRC 1024ES confocal laser scanning mi-

Fragment assembly and amino acid sequence prediction were performed using the GCG WISCONSIN PACKAGE version 10.1. Comparison of the BoTAP2 amino acid sequence with other available mammalian TAP2 sequences was performed using the GAP program from the GCG PACKAGE version 10.1 with a gap weight of 8. The secondary structure of BoTAP2 was predicted using the SOUSI program (http://sousi.proteome.bio.tuat.ac.jp). Multiple sequence alignment of mammalian TAP2 amino acid sequences was performed using ClustalW (http://dot.imgen. bcm.tmc.edu:9331/multi-align/Options/clustalw.html) followed by Boxshade 3.21 (http://www.ch.embnet.org/software/BOX_form. html). The phylogenetic analysis of TAP2 amino acid sequences was performed using the PHYLIP 3.5 program (http://evolution. genetics.washington.edu/phylip.html).

Results Cloning of full-length bovine TAP2 cDNA Based on partial BoTAP2 cDNA sequence in GenBank (accession number AF110317), two forward gene-specific primers (FP1 and FP2) and one reverse primer (RP1) were synthesized. Bovine PBMCs were selected in preference to epithelial cells such as MDBK, for isolation of RNA, because the former expressed higher levels of TAP transcripts. Isolated RNA was subjected to 3′RACE. Following two rounds of PCR amplifications (using FP1 and FP2, respectively), a specific DNA fragment of ~2000 bp could be observed. To verify the specificity, the 2-kb PCR product was subjected to Southern blot analysis (data not shown). The 2-kb 3′RACE product was cloned and sequenced. Based on the sequence data, three reverse primers (RP2, RP3, and RP4; Table 1) were designed and used in 5′RACE. The final 5′RACE DNA fragment of ~1000 bp was cloned and sequenced. Based on the 5′ and 3′RACE sequencing data, FP3 and RP5 primers (Table 1) were synthesized and used to amplify the full-length BoTAP2 cDNA. Analysis of BoTAP2 cDNA The BoTAP2 cDNA contains an open reading frame of 2160 bp encoding 719 amino acids. The lengths of the equivalent sequences in human, mouse, and rat are 703, 702, and 703 amino acids, respectively. BoTAP2 has a predicted molecular weight of Mr 79,000. Unlike human and mouse TAP2 sequences, BoTAP2 has four possible N-linked glycosylation sites, at amino acid positions 471, 504, 562, and 699. According to hydrophobicity plots, BoTAP2 consists of seven membrane-spanning domains, each containing 23 amino acids. The BoTAP2 peptide sequence has neither a potential leader peptide

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Fig. 1 Comparison of predicted amino acid sequence of BoTAP2 with available mammalian TAP2 amino acid sequences. Sequences represent HuTAP2 (human TAP2, GenBank M74447), GorTAP2 (gorilla TAP2, GenBank L49032), RatTAP2 (rat TAP2, GenBank X63854), MouTAP2 (mouse TAP2, GenBank M90459), HamTAP2 (hamster TAP2, GenBank AF001157), and BoTAP2 (GenBank AF318024). The darker the background of the character, the higher the degree of conservation. Uppercase letters in the consensus sequence indicate amino acid residues present in all the sequences compared, while lowercase letters indicate the most common amino acid among the six amino acid sequences compared. The Walker A and Walker B motifs are marked with solid and dotted lines, respectively

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Fig. 3a, b Induction of BoTAP2 mRNA following IFN-γ treatment. MDBK cells were either untreated or treated with 4 µg (per 5×106 cells) of recombinant bovine IFN-γ for 24 or 48 h. Cytoplasmic RNA was isolated, blotted, and probed with a 1.5-kb BoTAP2 probe. To confirm loading of equal amounts of RNA in each lane, the same membrane was stripped and reprobed with an 18S rRNA probe

Fig. 2 Phylogeny of BoTAP2. Phenogram showing the relationship of BoTAP2 to the other TAP2 sequences available. Predicted amino acid sequences of the TAP2 of bovine, mouse, hamster, gorilla, human, quail (GenBank AB007195), chicken (GenBank Al023516), rainbow trout (GenBank AF115537), salmon (GenBank Z83328), Xenopus (GenBank AF062387), and horned shark (GenBank AF108385), and hemolysin B (HlyB) of Escherichia coli (GenBank NP_052625) were compared using PHYLIP 3.5. HlyB of E. coli was used as the outgroup. Numbers on the branches refer to the bootstrap values recovered after 100 replications. HlyB of E. coli was selected because of its distant (prokaryotic ABC transporter), and close (more closely related than CFTRs and forms dimers as in TAP) relationship to TAP proteins. The tree is unrooted, i.e., the relationship does not consider the possibility that any one protein could be an intermediate in the evolution of another

nor ER retention signal. The NBD contains features diagnostic of an ABC-type transporter, the ABC signature sequence (LAVGQKQRLAIARAL), and Walker A and B motifs. The Walker A and B motifs of BoTAP2 are 100% identical to the corresponding sequences in human and gorilla TAP2. Comparison of the deduced BoTAP2 amino acid sequence to that of human, gorilla, mouse, rat, and hamster is shown in Fig. 1. BoTAP2 shows 80.6, 79.9,79.5, 79.4, and 79.2% homology to human, gorilla, mouse, hamster, and rat TAP2, respectively. As expected, phylogenetic analysis of BoTAP2 amino acid sequence with currently available TAP2 sequences revealed that BoTAP2 clusters together with its mammalian counterparts (Fig. 2). BoTAP2 mRNA expression TAP transcription can be up-regulated by IFN-β, IFN-γ and tumor necrosis factor (TNF)-α. Among these cyto-

Fig. 4 Differential expression of BoTAP2 mRNA in bovine tissues. Fifteen micrograms of total cellular RNA was size-fractionated on a 1% agarose/formaldehyde gel and transferred to nylon membrane. The blot was first hybridized with the 32P-labeled BoTAP2 probe, then stripped and rehybridized with 32P-labeled 18S rRNA probe. Band intensity was analyzed by PhosphorImager SF and the relative BoTAP2 expression was calculated as follows: intensity of BoTAP2 band/intensity of 18S band

35 Fig. 5a–d Intracellular localization of BoTAP2. COS-7 cells were transiently transfected with pEGFP-N1 (a, b) or pBoTAP2/EGFP (c, d). a In pEGFP-N1-transfected cells, EGFP expression was detected as a green signal (appears white in this figure) throughout the cell with the highest intensity in the nucleus. b, d The same cells were stained with an anticalreticulin antibody and the red signal (appears white in this figure) indicates location of the ER. c In pBOTAP2/EGFPtransfected cells, green fluorescence exhibited a typical ER distribution

kines, IFN-γ has the most profound effect. To study the level of expression of BoTAP2 mRNA in MDBK cells following bovine IFN-γ treatment, a Northern blot analysis was carried out (Fig. 3). As anticipated, a significant increase in BoTAP2 transcripts was detected 24 h following recombinant bovine IFN-γ treatment. Furthermore, the levels of BoTAP2 expression in different bovine tissues were examined by Northern blot analysis (Fig. 4). The same membranes were rehybridized with an 18S rRNA probe. To account for differences in sample loading, TAP2 expression was normalized to the amount of 18S rRNA in each sample. BoTAP2 transcripts were detected in a wide range of tissues with the highest degree of expression in jejunum and the lowest in liver. Intracellular localization of BoTAP2 We examined the intracellular localization of BoTAP2 by confocal microscopy of COS-7 cells transiently transfected with pBoTAP2/EGFP (Fig. 5). The cells transfected with pEGFP-N1 had the green fluorescence protein (GFP) distributed throughout the cell including the nuclei (Fig. 5a). In contrast, cells expressing the BoTAP2/EGFP

construct had the green fluorescence distributed only around the nuclei, yielding a classical ER pattern (Fig. 5c). The ER localization of BoTAP2/EGFP was confirmed by its nearly perfect co-localization with calreticulin (Fig. 5d).

Discussion Translocation of the cytosolic peptides generated by the proteasome into the lumen of the ER is an essential prerequisite for the assembly of the MHC class I-β2-peptide complex in the ER and subsequent transport to, and expression on, the cell surface. Hence this critical step of peptide translocation by TAP is targeted by several herpesviruses. Herpes simplex virus encodes an Mr 9000 immediate early protein ICP47, which binds to the TAP complex from the cytosolic side and inhibits its function (Ahn et al. 1996; Fruh et al. 1995; Hill et al. 1995; Tomazin et al. 1996). In contrast, the US6 gene product of human cytomegalovirus interferes with TAP function from the ER luminal side (Ahn et al. 1997; Hengel et al. 1996, 1997). Studies in our laboratory have revealed that BHV-1 and pseudorabies virus (Ambagala et al. 2000)

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also interfere with TAP function in bovine and pig epithelial cells, respectively. These findings prompted us to have a closer look at the bovine and pig TAP genes, their structure, and function. As the first step in this direction, we cloned, sequenced, and characterized the full-length BoTAP2. BoTAP2 encodes a polypeptide of 719 amino acids, with a predicted molecular weight of Mr 79,000. It shows the highest degree of homology (80%) to its primate counterparts. BoTAP2 has all the features unique to an ABC transporter including the ABC signature sequence, and Walker A and Walker B motifs. Walker A and B sequences in BoTAP2 are 100% homologous to those of the primate counterparts, at the protein level. Similar to the other TAP sequences, BoTAP2 does not have a cleavable signal sequence, a leader sequence, or an ER retention signal. Although extensive studies have been carried out on human and mouse TAP, the crystal structure of TAP is not yet available. However, based on hydrophobicity plots and the nature of binding of antibodies raised against different areas of TAP molecules, several topological models for human TAP1 and TAP2 have been proposed (Elliot 1997). According to the most recently proposed model, human TAP1 consists of eight transmembrane segments, and therefore both the N and C termini reside in the cytoplasm (Vos et al. 1999). In contrast, human TAP2 consists of only seven transmembrane segments resulting in the location of the N terminus in the ER lumen. According to our findings, BoTAP2, like human TAP2, also carries seven transmembrane segments. Therefore, we propose that BoTAP2 has a topology similar to that of human TAP2 (Fig. 6). Unlike human and mouse TAP2, the deduced amino acid sequence of BoTAP2 reveals four potential N-glycosylation sites. However, considering the possible locations of transmembrane domains, and ER versus cytosolic orientation of BoTAP2, none of these sites would be accessible for the N-glycosylation machinery. Therefore, these potential N-glycosylation sites in the BoTAP2 sequence most likely have no physiological relevance. Of note is that the human TAP1 sequence also has four potential N-glycosylation sites of which only one (position 279) is predicted to be luminally oriented and a potential target for N-glycosylation. However, the observation that human TAP1 is resistant to endoglycosidase-H and is unaffected by the glycosylation inhibitor tunicamycin (Elliott 1997; Russ et al. 1995) questions the physiological role of N-glycosylation in the TAP complex. Transcription of MHC genes can be up-regulated by cytokines such as IFN-β, TNF-α, and IFN-γ (Epperson et al. 1992). Of these three cytokines, IFN-γ has the most profound effect on TAP transcription (Elliott 1997). In the presence of stimulatory cytokines, human TAP1 transcription increases more rapidly than class I synthesis. This would ensure that, by the time class I expression reaches maximum levels, the supply of peptides to the ER would not be limiting. When compared to TAP1, TAP2 shows a delayed and less intense response to INF-γ

Fig. 6 Model for the topological organization of BoTAP2. The model shows seven (I–VII) transmembrane segments and two (Walker A and Walker B) ATP-binding motifs. The numbers indicate demarcations of amino acid positions in the transmembrane segments

treatment (Ma et al. 1997). To determine the levels of BoTAP2 transcripts in response to IFN-γ, we treated MDBK cells with recombinant bovine IFN-γ for 24 and 48 h, and the cytoplasmic RNA isolated was subjected to Northern blot analysis. We observed a significant increase in BoTAP2 transcripts at 24 h but the levels started to decline by 48 h. Higher levels of BoTAP2 gene expression are to be expected in tissues comprised of a large number of immunocytes. We examined several healthy bovine tissues for the level of expression of BoTAP2 transcripts by Northern blot analysis. The highest BoTAP2 expression was observed in jejunum which could be due to the presence of large numbers of Peyer's patches and trafficking lymphocytes. Bovine lung and spleen had considerable expression of BoTAP2 transcripts, while bovine liver had the least. The intracellular localization of the human TAP complex has been studied by both high-resolution immunoelectron microscopy and confocal microscopy. Using affinity-purified rabbit antibodies raised against the C terminus of human TAP1, Kleijmeer and co-workers (1992) demonstrated that the TAP complex is located in the ER and cis-Golgi by immunoelectron microscopy. In a later study, Russ and co-workers (1995), using confocal microscopy, confirmed that the human TAP complex is localized in the ER. According to our knowledge, anti-BoTAP2 antibody is not available. In our hands, none of the antibodies developed against the TAP of other species cross-reacted with BoTAP. GFP has proved to be a dependable and nondestructive tag which can be detected rapidly and easily (Hill et al. 1999; Htun et al. 1996; Niswender et al. 1995; Stauber and Pavlakis 1998). EGFP is an enhanced (35 times more fluorescence than wild type) and codon-optimized (for maximum expression in mammalian cells) version of wildtype GFP. As a result, EGFP has been used extensively as a tag to study the subcellular localization and trafficking of the proteins of interest (Jagiello et al. 2000; Valgardsdottir et al. 2001). Therefore, to examine the in-

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tracellular localization of BoTAP2, we constructed a BoTAP2/EGFP fusion protein. Cells transfected with pEGFP-N1 exhibited green fluorescence throughout the cell with the highest intensity in the nucleus (Fig. 5a). In contrast, BoTAP2/EGFP fusion protein yielded a classical ER pattern (Fig. 5c). This was further confirmed by co-localization of BoTAP/EGFP with calreticulin, a soluble ER resident protein (Fig. 5d). In summary, we cloned, sequenced, and characterized BoTAP2, and examined its expression in various bovine tissues. Using BoTAP2/EGFP fusion protein, the ER localization of BoTAP2 was also confirmed. Acknowledgements This article is published as Agricultural Research Division Journal Series No. 13477, with the approval of the University of Nebraska Agriculture Research Division. We thank Dr. Joe Zhou and Michelle Mathiesen at the Microscopy Core Facility of the Center for Biotechnology at the University of Nebraska-Lincoln, Neb., for their assistance with confocal microscopy. All the experiments conducted in this study comply with the current laws of the United States.

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