Biochimica et Biophysica Acta, 1216 (1993) 321-324 321 Elsevier Science Publishers B.V.

BBAEXP 90585 Short Sequence-Paper

Cloning a cDNA from human N K / T cells which codes for an unusual leucine zipper containing protein

Brian Dixon, Bichara Sahely, Lin Liu and Bill Pohajdak *

Department of Biology, Dalhousie UniL,ersity, Halifax, Not~a Scotia, B3H 4J1 (Canada)

(Received 7 June 1993)

Key words: Subtractive hybridization; cDNA cloning; Natural killer cell; T cell; Leucine zipper; (Human)

A 1724 base pair (bp) clone, B3-1, was obtained from a human NK subtracted cDNA library and sequenced. The cDNA encoded a 324 amino acid protein with a calculated molecular mass of 36 kDa. The deduced protein did not contain any hydrophobic domains, suggesting that it was not secreted or membrane bound. Extensive database searches showed no significant overall homology to any known proteins or genes. The protein did, however, contain an unusually long (twice normal) leucine zipper and the nuclear targeting sequence found in many transcription factors and oncogenes. The cDNA also contained three repeats of the sequence 'AT-ffI'A' in its 3' untranslated region, a motif associated with many oncogenes, transcription factors and interleukins. The mRNA for this gene is weakly expressed in resting NK/T cells.

Natural killer (NK) cells are lymphocytes that are able to destroy a wide range of target cells without prior sensitization (reviewed in Ref. 1). One approach to studying NK-cell mediated cytotoxicity is the isola- tion of genes that are NK cell specific. These cell-type specific genes often code for developmentally and functionally important proteins which specialize the activity of the cell. In order to increase the probability of identifying differentially T or NK expressed genes, especially those expressed in low abundance, subtrac- tive hybridization is often used [2-5]. We used the subtractive hybridization technique to isolate cDNA clones that are preferentially expressed in NK cells. One of the isolated cDNAs (B2-1) had a deduced protein sequence which showed a high degree of ho- mology to yeast protein SEC7 [6]. Another clone, B3-1, was isolated and used as a probe for Northern blots. Initial results indicated that both populations of purified NK and T cells weakly express B3-1 (unpub- lished data) and therefore were pooled in future analy- sis. Northern analysis of B3-1 expression in lympho- cytes stimulated with either P H A / P M A or IL2 re- vealed a slight increase following stimulation (25% and 2-fold increase in mRNA content, respectively) (Fig.

* Corresponding author. Fax: + 1 (902) 4943736. The sequence data reported in this paper have been submitted to the EMBL/GenBank Data Libraries under the accession number L06633.

1A). These values were obtained by subtracting the signal intensity of bands obtained using a control, non-induced gene (pHcGAP; Fig. 1B) to control for both loading and transfer efficiency. The pHcGAP probe recognises a message expressed in the stimulated Jurkat control lane (Lane 1, Fig. 1B), but the B3-1 probe does not recognise a message in that lane (Fig. 1A). The NK subtracted library was constructed by subtracting messages expressed in the T helper cell line Jurkat. Preliminary results using a panel of 11 cell lines suggest that B3-1 is lymphoid specific (unpublished data).

The complete (both strands) sequence of B3-1 (clone 6) is shown in Fig. 2. The 1724 bp insert con- tains a 972 bp open reading frame which starts at nucleotide position 92 encoding a putative protein of 324 amino acids and a molecular mass of 36 kDa (Fig. 2). The size of the cDNA is close to that estimated by Northern analysis (1.9 kb), however size estimations from Northern blots are not very accurate. An analysis of the 3' untranslated end of B3-1 revealed that there are three A TTTA sequences (Fig. 2, underlined) and a polyadenylation signal (Fig. 2, bold). These ATTTA regions have been found predominantly in the 3' un- translated ends of the mRNAs of several proto-onco- genes and lymphokines and are responsible for the very low levels of these proteins [7,8]. While the start codon for the open reading frame indicated in Fig. 2 has an A codon at the - 3 position (i.e., three bases upstream), it does not reside in a perfect Kozak con-

322

sensus sequence [9]. Since the untranslated sequence upstream from this start codon does not contain any in-frame stop codons, it is possible that this clone is not full length and that the correct start codon is not present in this cDNA clone. Thus, the predicted pro- tein may be lacking a portion of its N-terminus.

Extensive database searches for homology to B3-1 were performed. Overall, no other known genes or proteins showed a high degree of sequence homology to B3-1. Using the PROSITE GCG program [10] consensus sequences were identified in the deduced amino acid sequence. A reduced stringency search, allowing for one mismatch, revealed several sequences which match the leucine-zipper motif [11] (Fig. 3). The leucine zipper region is located between residues 132 and 188, and contains several possible leucine zippers (Figs. 3 and 4A). One possibility is that there are two standard leucine zipper sequences back-to-back from positions 132 to 160, then 160 to 188. Both of these zippers would contain five repeats of the LxxxxxxL motif with allowable substitutions which are found in other zipper proteins [12] (Fig. 3). The best match to the leucine zipper consensus sequence is found be- tween amino acid residues 139 and 167. This region has five repeats of the L motif with a glutamic acid residue following the first L (Fig. 3). This residue is usually glutamic acid or glutamine [13]. In addition,

A

28S~

18S~

B

28S~.

18S~

1 2 3 4 5

Fig. 1. Northern blots showing the stimulation of B 3 - 1 expression by P H A / P M A and IL-2. Lane 1, Jurkat control RNA. Lane 2, RNA from unst imulated cells. Lane 3, IL-2 stimulated cell RNA. Lane 4, unstimulated cell RNA. Lane 5, P H A / P M A stimulated cell RNA.

The position of the 18S and 28S rRNA bands are indicated.

there is an asparagine residue 18 amino acids upstream from this putative zipper which is surrounded by sev- eral basic amino acid residues (Fig. 3), another con- served feature of most leucine zippers [13]. There appear to be two important requirements to form a leucine-zipper. First, there must be four or five leucines repeated every seventh amino acid residue (reviewed in Refs. 11,12,14). Second, and more importantly, the region containing the stretch of amino acid must form an a-helix [11]. The predicted [15] a-helix stretch of 53 residues from positions 133-189 contains two of the leucine zippers (Figs. 3 and 4). When searching for regions of homology between B3-1 and c-los or c-myc (which both contain leucine-zipper sequences), both sequences aligned with that of B3-1 such that the leucine 'zippers' (given the mismatches in B3-1) coin- cided exactly with each other (unpublished data). There is also a region of high proline content from residues 191 to 197 (Fig. 1). This too is a feature of many leucine zipper sequences [16,17].

The hydrophobicity profile of the inferred amino acid sequence was determined [18] (Fig. 4B). No signif- icant hydrophobic regions resembling ER-targeted leader sequences [19] or transmembrane domains [20] were found.

It is presently not known if B3-1 encodes a DNA- binding protein or a negative regulator of other leucine zippers, like the protein Id [21]. A conspicuous feature of B3-1 is the presence of four consecutive negatively charged glutamate residues near the carboxyl-terminus in a region that appears to form an amphipathic-helix (Figs. 3 and 4). It is of interest to note that clusters of negatively charged amino acids are also found in many transcriptional activation factors [22,23] and that sev- eral of the activating sequences studied have been found to form amphipathic-helices [22]. These nega- tively charged regions are believed to activate tran- scription by interacting directly with a hydrophilic re- gion in RNA polymerase II [23]. A potential nuclear targeting sequence, RKSRK, is located from amino acid residues 295-299. Southern analysis showed that the B3-1 gene is single copy (unpublished data).

There are several indications that suggest B3-1 might be a putative nuclear transcription related fac- tor. The 3' untranslated end of B3-1 cDNA clone has three A TTTA sequences that are also found in several transcription factors and proto-oncogenes. The weak expression of B3-1 in several cell lines supports the short half-life of this mRNA. Also, the protein has a potential nuclear targeting sequences, two possible leucine zipper motifs and a potential transcriptional activation domain (acidic clusters). Since B3-1 has features indicative of a transcription activator, B3-1 may belong to the new class of non-DNA binding transcription factors. This possibility is presently being investigated.

323

1 GCTCCTGCAACACAGC

17 AGCAATGGCAATTTGGCGGACTTCTGCGCTGGGCCAGCGTATAGCTCTTACTCCACACTCACCGGCAGCCTTACG

92 ATGAACGATAATAGAAGGATTCAAATGCTAGCAGACACGGTGGCTACTCTGCCTCGGGGACGAAAGCAGCTTGCT

M N D N R R I Q M L A D T V A T L P R G R K Q L A 25

167 TTGACCAGATCAAGTTCTTTAAGTGACTTTTCCTGGTCTCAAAGAAAGCTTGTTACTGTGGAGAAGCAGGATAAT

L T R S S S L S D F S W S Q R K L V T V E K Q D N 50

242 GAAACATTTGGATTTGAAATTCAGTCTTACAGGCCCCAGAATCAGAATGCCTGCTCCTCGGAAATGTTCACTTTG

E T F G F E I Q S Y R P Q N Q N A C S S E M F T L 75

317 ATATGCAAAATACAGGAGGACAGCCCAGCTCACTGTGCTGGCCTGCAAGCTGGTGATGTCCTTGCAAATATCAAT

I C K I Q E D S P A H C A G L Q A G D V L A N I N I00

392 GGTGTGAGCACAGAAGGTTTTACCTACAAACAAGTCGTTGACCTGATCAGATCGTCCGGAAACCTGCTAACGATA

G V S T E G F T Y K Q V V D L I R S S G N L L T I 125

467 GAGACTCTTAATGGAACAATGATTCTGAAAAGAACGGAGCTTGAAGCAAAGCTGCAGGTTTTAAAGCAAACTTTG

E T L N G T M I L K R T E L E A K L Q V L K Q T L 150

542 AAACAAAAATGGGTGGAGTACAGATCTCTGCAGTTACAGGAACATCGTCTGCTTCATGGTGATGCAGCTAATTGC

K Q K W V E Y R S L Q L Q E H R L L H G D A A N C 175

617 CCCAGTTTGGAAAACATGGACTTGGATGAATTGTCTTTGTTTGGACCCCTGCCTGGGCCAGGCCCAGCCCTTGTG

P S L E N M D L D E L S L F G P L P G P G P A L V 200

692 GACCGGAATCGATTATCCAGTGAGAGCAGCTGTAAGAGCTGGCTGAGCTCCATGACGATGGACAGTGAAGATGGC

D R N R L S S E S S C K S W L S S M T M D S E D G 225

767 TACCAGACGTGTGTGTCTGAGGACTCCAGCAGGGGTGCCTTCAGTCGGCAGCAGAGTACAGATGATGAGTGCTTT

Y Q T C V S E D S S R G A F S R Q Q S T D D E C F 250

842 ATCCCCAAGGAGGGGGATGATTTTCTGAGGAGGTCATCTTCAAGGAGGAACCGGAGCATCAGTAACACCAGCAGC

I P K E G D D F L R R S S S R R N R S I S N T S S 275

917 GGATCCATGTCTCCCTTGTGGGAGGGCAACTTATCAAGCATGTTTGGGACCCTGCCCCGGAAGAGCAGAAAGGGA

G S M S P L W E G N L S S M F G T L P R K S R K G 300

992 AGTGTCCGAAGAGTTGCTTTGAAATTTATCCCTGGCCTTCATCGTGCTGTGGAAGAGGAAGAAAGTCGCTTTTGA

S V R K Q L L K F I P G L H R A V E E E E S R F * 324

1067 CGGATTGTGGTGTCCTTTCAAATTAGCTTATTTCACAAATATCTCTAGACTCACCCAGATCCCAGCTTGGTGGGA

1142 AAGTGCAGAAGAATTGCAAAACTGACATCCCATTTCACAGCAATAGTGACCTTTATTTAAATTGTTGTGTTATAG

1217 TTTATGCTTCTTAAATCATTTTTCAACCTAAACAGCCAATTTCTAAGCAGACAGGAAAACTAAATAATAAGTTAA

1292 TTAATATAACAAAGATGCAGGTTCCTGCTCATTCCAGTAATGTCTTTGAAAGCAAAACTAATATTTATTTTCTAG

1367 ATTATCCCTGTGAATAATTGAGAACTTTTTGGAGTCAAGTATGAATAAAGGTGTGGCAGAATATAATAATCTGGA

1442 CTATTTTCTATAGGATAATTGCTGGGTTATAAAATCTTAGGTTTGCTTATGCCCAGTAGCTCCTGCGGAGGCTTA

1517 ATAATAGGCAATTTTGAATTTGTTCAAACCTGTAATGGCTTGTAAACAAAGATGACCATCAGCTGTTTCTCACAT

1592 CTATAGTGACAATAAAGCGGGAAGTATAAGATTTAATAGGAGGGGTTAAGGTTCATGAGAACCATGGAAAGATGT

1667 GGTCTGAGATGGGTGCTGCAAAGATCATAATAAAGTCATTTTTATAGACAGTCTAAACAAAAAAAAAAAAAAAA

Fig. 2. Nucleotide and deduced amino acid sequence of clone 6. The polyadenylation signal sequence is in bold type. The A T T T A sequences in the 3' untranslated region are underlined. Double-s t randed D N A template was obtained using the alkaline lysis method [24] and sequenced

using the d i d e o ~ chain-termination method [25].

C/EBP VETQQKVLELTSDNDRLRKRVEQLSRELDTLRG - 341

JUN ARLE E KVKTLKAQNS E LAS TANMLTE QVAQLKQ -320 A LX6 LX6 NX6 MX6L L-MYC QALVGAEKRMATEKRQLRCRQQQLQKRIAYLSG -363 Leuoi.¢ 7i~,

FOS DTLQAE TDQLE DKKSALQTE IANLLKEKEKLE F -296 A~o N k T~A YAP1 KELEKKVQSLESIQQQNEVEATFLRDQLITLVN -123 s -,~ lii::iiiiiiiii!{!i!iiil ~.~.~"k~l II i"--'- ~'

/ \ / I /.,~> t \ / \ CREB KCLENRVAVLENQNKTLIEELKALKDLYCHKSD -342 B n~,i~ Region Leucine zipper P,oline Nuclear ATTTA

HBPI EELGQRAEALKSENSSLRIELDRIKKEYEELLS -239 r~:%Ix 6ix 6 KX6L n,~e~i.~ x3

Consensus --L* ..... L ...... L ...... L ...... L-- -i_z° I|II~ i.I[ 1 I" I r" I I~I... iiiii~

B3-1 IST GTMILKRTELEAKLQVLKQTLKQKWVEYRSLQL -162 -31 .... i .... l .... i .... I , , i t I , , I B3-1 2ND RSLQLQEHRLLHGDAANCPSLENMDLDELSLFG -190

B3-1 BEST TELEAKLQVLKQTLKQKWVEYRSLQLQEHRLLH -169

Fig. 3. Al ignment obtained when B3-1 amino acid sequence was compared with that of other leucine-zipper containing proteins. Numbers represent position of the last residue in each protein sequence. * = E or Q. The sequence of L-myc is from Ref. 12, the

rest of the zipper sequences were obtained from Ref. 13.

C Alpha Helix . . . . . 100 200 300

Fig. 4. (A) Schematic diagram of the t73-1 gene indicating the important D N A or derived amino acid sequences. (B) Hydrophobic- ity plot [18] of B3-1 derived amino acid sequence. To scale with the diagram in A. (C) Predicted secondary structure [15] of B3-1

derived amino acid sequence, also to scale.

324

We thank Dr. R. Perlmutter for the peripheral T cell cDNA library. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.

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