Gene, 86 (1990) 285-289 Elsevier

285

GENE 03383

Cloning and sequence analysis of the human gene encoding

eosinophil major basic

protein

(Allergy; Alu repeat; gene polymorphism; hypersensitivity; promoter; pro-protein; recombinant DNA; toxin)

Robert L. Barker, David A. Loegering, Ken C. Arakawa, Larry R. Pease and Gerald J. Gleieh Departments of Immunology and Medicine, Allergic Diseases Research Laboratory, Mayo Medical School. and Mayo C ~ Rochester. MN 53903 (U.S.A.) Tel. (507) 284-8333

and Foundation.

Received by D,M, Skinner: 7 July 1989 Revised: 10 October 1989 Accepted: 12 October 1989

SUMMARY

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and mammalian cells, is a single polypeptide rich in arglnine. The gene, mbp, was cloned and its nucleotide sequence determined. The 3.3-kb gene consists of six exons and five introns, one of which contains an Alu family repeat. The combined exon sequence is similar to previously reported mbp cDNA sequences. The gene is immediately preceded by a putative promoter containing typical TATA and CCAAT boxes. Southern blots indicate that mbp exhibits limited polymorphism.

INTRODUCTION

Eosinophil granule major basic protein (MBP) comprises the crystalloid core of the eosinophil granule (Lewis et al., 1978) and is a 13.8-kDa single polypeptide rich in arglnine (Wasmoen et al., 1988). MBP is a potent toxin for helminths and mammalian cells in vitro and has been localized on damaged helminths and tissues in hypersensitivity diseases including bronchial asthma (Gleich and Adolphson, 1986). A molecule indistinguishable from MBP which does not appear to be produced by eosinophil (Wasmoen et al., 1990) has been localized to placental X cells and placentalsite giant cells (Maddox et al., 1984) and increases in materhal plasma just before labor (Wasmoen et al., 1987).

The mbp cDNA from the promyelocytic cell line HL-60 which produces MBP indicates that MBP is translated as a slightly acidic pre-pro-protein with an acidic pro-portion (Barker et al., 1988). This acidic pro-portion may mask the toxic effects of mature MBP and protect the eosinophil from damage while the protein is processed through the endoplasmic reticulum to its sequestered site in the eosinophil granule as toxic MBP (Barker et al., 1988). Restriction analysis and Southern blot hybridization of human genomic DNA suggested that mbp is a single copy gene (McGrogan et al., 1988). Here we report the structure of mbp.

EXPERIMENTAL AND DISCUSSION

Correspondence to: Dr. R.L. Barker, Department of Immunology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905 (U.S.A.) Tel. (507) 284-5959; Fax (507) 284-1637. Abbreviations: aa, amino acid(s); bp, base pair(s); kb, kilobase(s) or 1000 bp; MBP, major basic protein; mbp, gene encoding MBP; oligo, oligodeoxydbonucleotide; nt, nucleotide(s); SDS, sodium dodecyl sulfate; SSPE, 0.18 M NaCI/10 mM NaH2POJI.0 mM EDTA pH 7.4; ter, termination codon. 0378-1119/90/$03.50 © 1990 Elsevier Science Publishers B.V.(Biomedical Division)

(a) Cloning of mbp A tCharon4A human fetal liver genomic library (ATCC 37333) containing 1 x 10e independent recombinants with an insert size of 15-20 kb was screened with mbp cDNA (Barker et al., 1988) essentially as previously described (Maniatis et al., 1978). Phage were sequentially adsorbed

286 onto 2-8 × 8 cm nylon membrane filters per each library plate. Both sets of filters were prehybridized in 5 × $SPE/50~o formamide/5 × Denhardt's solution (0.02~o Ficoll/0.02% polyvinylpyrrolidone/0.02% BSA)/0.5% SDS and 0.005% denatured salmon sperm DNA for 2 h at 42°C with eight filters per 50ml prehybridization fluid per bag. Filters were hybridized with 1.0 ng of labeled mbp eDNA per ml of fresh prehybridization fluid containing 10~o dextran sulfate and 2 × Denhardt's solution overnight at 42°C. The mbp eDNA was oligolabeled (Feinberg and Vogeistein, 1983) with [0c-32P]dCTP and purified over Sephadex G-50 spun-columns. Filters were washed twice at room temperature for 15 min in one liter 2 × SSPE and 0.2% SDS per 40 filters followed by two 15-min 50°C washes in 0.1 × SSPE and 0.2~ SDS, slightly air-dried and exposed to Kodak XAR5 film with intensifying screens for 48 h at -70°C. After an initial screen of 480 000 pfu, two clones positive to the mbp cDNA were selected from the library and plaque purified. Plaque hybridization of the two mbp clones with oligos (Barker et al., 1988) generated from the mbp cDNA sequence suggested that only one clone contained a complete copy of mbp. Recombinant phage DNA from this clone was purified (Yamamoto et al., 1970) and restricted with BamHl + EcoRl. Southern blots (Southern, 1975; Botchan et al., 1976) of these restriction fragments from the 16-kb insert hybridized with mbp cDNA and oligos identified a 2.8..I:b BamHI fragment which contained most of the mbp and ~ 3.8-kb EcoRl fragment which contained the remaining ~.' portion of the gene and overlapped 1,4-kb of the 2.8-k~ BamHI fragment. The EcoRl fragment was restricted with BamHl which resulted in 2.4- and 1.4-kb fragments. The 2.4-kb EcoRI-BamHl fragment and the 2.8-kb BamHl fragment were ligated into M13mpl0 and Ml3mpll and sequenced by the method of Sanger et al. (1977) using [3SS]dATP (Biggin et al., 1983).

(b) Sequence analysis of mbp The nt sequence of mbp together with 5' and 3' flanking regions is shown in Fig. I and a restriction map of the gene based on this sequence is presented in Fig. 2. The gene is organized into six exons separated by five introns. In general, the first and second exons comprise the 5' untranslated region and the putative MBP signal peptide while the third exon codes the pro-portion and beginning of mature MBP. The three remaining exons code the rest of MBP and the 3' untranslated region. Since the three dimensional structure of MBP is unknown, it is not possible to determine ifexons 2-6 represent compact polypeptide structural units (Go, 1981). The nt sequence of the exons is identical to the mbp

eDNA sequence determined by Barker et al. (1988), except that exon 1 begins with three A's while the cDNA begins with four A's. The reported eDNA sequence (Barker et al., 1988) has a typographical error at position 318 (Fig. 1, nt 1525) which should be G instead of C. The 5' flanking sequence (Fig. 1) immediately preceding the mbp sequence equivalent to this eDNA has putative promoter elements at expected positions (see section e below). This conf'~nns the results of previously reported primer extension reactions (Barker et al., 1988) that the mbp eDNA found is a fulllength copy of the mbp message. The nt sequence ofthe exons is similar to the mbp eDNA of McGrogan et al. (1988) which also was isolated from HL-60 cells. This eDNA contains two typographical errors (M. McGrogan, personal communication). First, nt GA at positions 731-732 (Fig. 1, nt 3189-3190) are transposed (the correct nt at these positions are AG). Second, the nt GC should be inserted between the A at position 759 (Fig. 1, nt 3217) and the T at position 760 (Fig. 1, nt 3220). The corrected eDNA of McGrogan et al. (1988) lacks the first 19 nt of exon 1 and differs only at position 719 (T in place of C) in the coding region of mbp which does not change the leucine coded at this position. This eDNA also differs at one position in the 3' untranslated region of mbp. The eDNA has a G inserted between nt 3220 and 3221 of

mbp. The intron-exon boundaries were identified by comparing the gene sequence to mbp eDNA (Barker et al., 1988) and following the GT-AG rule (Breathnach et al., 1978). The introns are inserted either between codons or within codons between the first and second nt of a triplet. Intron 4 contains a copy of a human Alu family repeat (Fig. 1). This Aiu repeat is 64% identical to the human Alu genomic consensus sequence ($chmid and Shen, 1985). The average divergence of individual cloned members of the human Aiu family from the genomic consensus is 14~o (Schmid and Shen, 1985). Although some Alu clones vary as much as 23% from a consensus sequence (Deininger et al., 1981), we are not aware of any other human Alu family member that varies as much from the genomic consensus sequence as that reported here. No other type of repeat was found in the mbp intron structure.

(e) 5' flanking sequence The area immediately preceding mbp (Fig. 1) contains elements of a typical eukaryotic promoter. A putative TATA box which is identical to the TATA consensus sequence (Corden et ai., 1980) begins at nt -28 and a putative upstream promoter element which is similar to the CCAAT box consensus sequence (Benoist et al., 1980) begins at nt -69. No known enhancer motifs were found in either orientation in the 5' flanking sequence or down-

287 5" f t m k i n 8 SeClUm:e - 292 TTCAAAACACCAGTAAAACAGGGGAGATATGTATTTTGGAAAAGCACCCAAG -2/,0 G~GATTCTGAAGT~TAGCCCAGGATAAGAACCATTGCCCAGAG~TGTT~CAGATG~CCCCTGGGTTCCT~AAGTGGGTATCGGGAGAGAAATCTTCACTGAAT~TGAGTG~T~ -120 AGGGAA6T•ATGAAAT•GTCCTTATCA••CTT•CTATCTC•CT•TGACAGA•GCAAACT•TCTCTC•CT••6•GAAGTT•CTCCAAGGCCTCTATATAAGAA6TCTTT6TGAGA•GAAGC Exon 1 lntrm 1 1 A /tAG /LAG GAC CT6 GGC TTT GGG AAG ATC TAA AGA CCC AGG AAG GTC TCT GCG TGG GTGAGTGCTTTCTCTGCTGTGGTGGACCTGGTGACAGTTTATTC ter 100 TCCCAGGAGGTCCCTGGCTGTGGCTGACAGTTTCT~GAGGGCTGGCA~G~GTCTACCTGT~GCTTTCAGGTTATGAGGAT~TCAGCAGGGGCAGCCTTCATCCTCTGCCTT~CATT~ 220 TTCT6CGGGATGTGAAA6TGCTCCTT~CT~GGGAAAG~AGATGGT~GAGACAT6GA~GAGG~T~T~T~CTTCTTGAACTCT~AGGAGGGGACATA~TT¢T~GT~TATGT~TTC 340 CTAGGAAAGCCAATAATCATTGCTTCTCCCGCCT TTTTTATGTCATAGACTCTGAGGGACCCATTAAGTACAAAGAAATAAGCGTAATAGTCCCTTCTTTACTTCCGGGCCT~ 460 GCCAGCCTCAGCCAC¢CCTCAGGGTT TGCTGCGTTCTGTTTAGAAAGAGGTCCTTGCGTCCTGGv'TcCIrGGAGCATCAGGAGCTGGGCTTGGCATGAGCTTTTCTGGCCCATCCTGAT I 578 TTCTATTCAGGCCTTCTTTTTCTCCA~CTCACTCCCACGGTCCCCTAAT~GTGT~ATTGTGATGT~TGT~CAT~TGTGTCTGTGTGT~TCAATGA~-~`CTGTGTTCTCCGTTGCA~ Exon 2 IntNn 2 695 CAT AM GCC /tAG ATG AAA CTC CCC CTA CTT CTG GCT CTT CTA TTT GGG GCA GTT TCT GCT CTT CAT CTA A GTAAGTGTTTTTTGCCTTCAGTCTTT -15 m k L p I L t a t t f 9 a v s ~a t h t 791 CTTTCTCTGTTTTTTCCCTTTCTAT•GTAGATGGGGTCAGAGTTACACACCCACCCCCTTCTTT•ATCGTCTTCTATTTCTGNkTTTCT•TGTGCTT/L4AGGGATGGGGACTCTATGGCC 911 A•GA•TTGAAAGGATTTcTCAA•GcGTcTGTTATGT•TGTGGTcTTGGTTcTAcTGTGACATTCcCAATTTT•TccTTTcTcCATTAT•cTTA•TTTGA••TTAcTGAGT•••TTcTcTc 1931 CTTTAACTCTCTTAGCATCGCCAT~AAGTAGGTGGTATTGTATAcCCATTTCACAGAAATACA~CTGGTGGATGATGGAAcCA~TAC~CAAGCCCATGACT6CcCGAcTCTAAGTCCATG 1151 CTCTTAACCA•CTTGACCTTGTCAGGCAGCTTGGGTTCCCCTCATAGAGACTGGGTTCCAGGTTC•CCTTC•CAGGCAGAGTTGAGCA•T•TGAT•••CAGGGCAAGGTGTGAGCT•T•T F.xon 3 1271 GTGGTTCTGGGGAGGAACAAGGGGAGATGTGAAGGAAGGACACTTAGCTATCCTCCCTGCCAGGG TCT GAG ACT TCC ACC TTT GAG Ace CCT TTG GGT GCT AAG 5

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AGT GAit GAT GCC TCC /LAG AM GAT GGG GCT GTT GAG TCT ATC TCA GTG CCA GAT ATG GTG GAC AM AAC CTT ACG TGT CCT GAG GAA GAG s • d a s k k d 9 a v • s | s v p d m v d k n t t c p • • • GAC ACA GTA AM GTG GTG GGC ATC CCT GGG TGC CAG ACC TGC CGC TAC CTC CTG GTG AGA ACT CTT rAG ACG TTT ACT CAA OCT TGG d t v k v v g i P 9 c q ii~i c r y t t v r s | q t f s q a u lntron 3 1642 GTGAGTGG~CTAT~CTGAG~CTGAGGTGGGAGCATGGAACGGGTGTGGGATATG~C~CAGCATT6~TATCA~TGG~T~TTTTT~CATTGAGGG~TGG~GGTGTcAGTAGAA~TG 1762 A••cTCAGAGAGGTGTTG••GTAAGAGGGGAGG••CAc•TA•AAACAGAAGTTGCATTTTGGT•T•cMc•TT•AAAT0GTTGTGGCAG•GGA••GAG•GAAT6AAf TGTGGGGACTGAA Exon4 1882 GACCCATGTGAATTCATGTAGGAAGGATGCTCC&TTCTTTGTCTTTTATCCTGCCCTGTAG TTT ACT TGC eGG AGG TGC TAC AGG GGC AAC CTG GTT TCC ATC 108 f t C r r c y r g n t v s i 1985 CAC AAC TTC AAT ATT AAT TAT CGA ATC CAG TGT TCT 6TC AGC 6CG CTC AAC CAG 6GT CAA GTC TGG ATT GGA 6GC AGG ATC ACA GGC TCG 122 h n f n i n y r i q c s v s a | n q g q v u i 9 g r | t g s Intron/~ 2075 GTAA6A•AAGT6T6AA•ACTAAATG•GGTG•A•CTGCT6ATCTCAGc•A6GACT•AG•TTG•AT•AGATTT•T•TG•TTTT•T•cT6TATAAT•T••A6AA•AAc•AGGGATAGATGGAc 2195 A~¢CA¢~f`~cAA~A~TGAGG~G~TG~cTG~GCATTCA~GGAAGAGcTAAGGATTTAGAATCA0GAGGTTTG~GTc~AAGTTcc~TTcCAT~TcTGAcTAT~TATGTAAcTTAA~TTA~c 2315 TGGGCAT•GT•GTGCAT•T•T•TAATC•TAGCTA•TTGGGAGG•TGAGGCAGGAGAGT¢A•TGGAA••T•GGAGA•AGAGGTTGCGGTGAQC•GAGAT•GAGCCATTGCA•T•CAG••TG 2435 GGc~A~GA~CGN~T~C~TC~AAAATAA/~TAA~TAAATAAATAAAATAAAAAAAAATTAAAACAAGACCATGA~TTT~TTTC~TCAT~TCTAGGATGA~TT~G¢AA~C~TT~TTC 2535 TA•CTTTT•TTAGGGCTGGAAGGACAAGCCTGTCACT••GAT•CATAGAATCTGATGGTGATAATT•CCGT•GATCAGCATTTCAGAT6A•TA•GACAGTT•cCATCATG•T•CA•CAGG Exm S 2675 GAAGGGCCCATTGCC••GTGGGcAGcAGAAAGAGCTGGCAGATA•GGGG•CAGGTc•GCTTcT•TG••TT•cCTCTGc•••ATc••TT•TT•c•CTc•TGCTT••TC•AG GGT CGC 152 0 r 2791 TGC AGA CGC TTT CAG TGG GTT GAC 6GC AGC CGC TGG AAC TTT GCG TAC TGG GCT GeT CAC CAG CCC TGG TCC CGC GGT 6GT CAC TGC GTG 154 c r r f q w v d 0 s r w n f a y ~ e e h q p ~ s r g 0 h c v Intron 5 2881 GCC CTO TOT ACC CGA G GTGAGGTGGGG•TGGGGATGAA•GATGGAAAGGTCTGGGAGATGGGAAGTG•CccAAGGAGGAGATGCTA•AAAGAGCCTGA•c•TTTGTGGGAGAGG 184 e t c t r 2995 CTT••TG••TCTTTTATATACT•T6AcTCCACAGCAGT•TGT••GTGGGAAAAGAGG•CcT•cT6T••GTTGA•TT•••AT•GACAAGAGGcT6AAA•TcccTTTcT•TTcT•CcTTCA• 3115 AO

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Fig. I. Nucleotide sequence ofhuman

-292 to -3316 and aa -15 to -207 are numbered on the left o f the figure. The C C A A T box, T A T A box,

eukaryotic initiation signal, polyadenyiation signal and poly (A)-addition site in eDNA are double underscored. An arrow in the -1 $ line indicates putative signal peptide cleavage site. A shaded box surrounds first aa in eosinophil granule MBP. Alu repeat is underscored. BomHI recognition sites are italicized. Both strands of each of the 2.8-kb BamHl restriction fragment and the 0.7-kb of the 2.4-kb EcoRI-BamH! fragment immediately preceding the 2.8-kb BamHl fragment which comprise this figure were sequenced in their entirety.

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Fig. 2. Restriction map of human mbp. Exons are boxed. Exon and intron nt lengths are indicated beneath box or line. Only useful restriction sites are shown.

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ACKNOWLEDGEMENTS

We thank Linda H. Arneson for preparation of the manuscript. This work was supported in part by grants from the National Institutes of Health (A109728, AI 07047, AI 00706, and A122420) and the Mayo Foundation.

REFERENCES

3.8 - ' ~ 2.8 " ~

Fig. 3. Southern blot ofmbp. Genomic DNA from four different individuals was restricted with EcoRI (lanes 1-4) or BamHl (lanes 5-8) and 10 ~g of sample DNA per lane was run on an 0.8% agarose gel. DNA. was blotted onto a nylon membrane and hybridized under standard conditions (identical to those used for library screening in section a) to 32P-labelled mbp eDNA. Lanes: I and 5, Japanese; 2 and 6, African (black); 3 and 7, Asian (Indian); 4 and 8, European (caucasian).

stream within introns. Since MBP appears to be produced by eosinophils, as well as other cell types (Wasmoen et al., 1990), it is likely that transcriptional control of mbp is regulated by additional elements which are present in the sequence reported here and/or flanking sequences.

(d) GenomleSouthernblots Human genomic DNA isolated by standard methods (Maniatis et al., 1982) from peripheral blood mononuclear cells from four different individuals was restricted with EcoRI or BamHl. Southern blots of this DNA hybridized with mbp eDNA (Fig. 3) identified 6.5-kb and 3.8-kb positive EcoRI bands and a 2.8-kb positive BamHl band. These bands were identical among all four individuals suggesting that mbp exhibits limited polymorphism. These results are similar to those of McGrogan et al. (1988) for genomic DNA from human fibroblasts and HL-60 cells. Although the BamHl Southern blots reported above show only a 2.8-kb (Fig. 1, nt 522-3316) band positive to mbpcDNA, a 3.8-kb BamHl fragment is present in the mbp clone which contains the remainder of mbp. This fragment may not appear on genomic BamHI restricted Southern blots hybridized with mbp eDNA, since only 55 nt of fulllength mbp eDNA are present in the 3.8-kb BamHI fragment which does not generate a sufficient signal under standard hybridization and washing conditions.

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sis of haman cosinophil granule major basic protein. J. Biol. Chem. 263 (1988) 12559-12563. Wasmoen, T.L., McKean, DJ., Benirschke, K. Coulam, C.B. and Gleich, GJ.: Evidence of cosinophil granule major basic protein in human placenta. J. Exp. Meal. 170 (1989) 2051-2063. Yamamoto, K.R., Alberts, B.M., Benzinger, R., Lawhome, L and Treiber, G.: Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40 (1970) 734-744.