Proc. Nat!. Acad. Sci. USA Vol. 89, pp. 1715-1719, March 1992 Biochemistry Isolation and sequence of the granulin precursor cDNA from human bone marrow reveals tandem cysteine-rich granulin domains (epithelin/growth factors/kidney/i ion) VIJAY BHANDARI, ROGER G. E. PALFREE, AND ANDREW BATEMAN* Endocrine Laboratory, Royal Victoria Hospital, Department of Medicine, McGill University, Montreal, PQ, H3A lAl, Canada Communicated by Seymour Lieberman, November 15, 1991 (receivedfor review September 17, 1991)

ABSTRACT Granulins are candidate growth factors re- mal (EGF) (14). Some ofthese peptides may be cently discovered in human and rat inflammatory leukocytes involved in initiating or sustaining an inflammatory response and bone marrow. Two granulin homologs, epithelin 1 and 2, (15, 16), in tissue remodeling at the site of a wound by occur in the rat kidney. Epithelin 1, which is probably identical regulating matrix formation (17, 18), and in the recruitment of to rat leukocyte granulin, exhibits proliferative and antipro- neighboring by chemotaxis (19). The similarity liferative effects on epithelial cells in vitro. Here we show by between the molecular biology of wound repair and tumor cDNA analysis that the prepropeptide for the human granulins development has been discussed (20), and it is likely that is a 593-residue glycoprotein, containing seven tandem repeats many of the same regulatory polypeptides are involved in of the 12-cysteine granulin domain. By Northern blot analysis, both processes. expression was seen in myelogenous leukemic cell lines of The association of human and rat granulins with inflam- promonocytic, promyelocytic, and proerythroid lineage, in matory leukocytes (7) and the mitogenic properties of epi- fibroblasts and was seen very strongly in epithelial cell lines. thelin 1 suggest roles in wound healing. The presence of Some epithelial cell lines respond to the mature peptide and epithelins in the kidney (8) argues for other functions inde- express the gene. Among tissues examined, the kidney had the pendent ofthe inflammatory response. The possibility exists, highest levels of granulin mRNA. however, that the epithelins are not intrinsic to the kidney but are present through a passive mechanism, such as leukocyte Extracts of cells elicited at sites of contain entrapment. Little is known about the cellular distribution of several low molecular weight peptides. Many of these are granulins and whether they are products of one or several involved in the destruction of pathogens (1). Some also have under coordinate or independent control. To address potentially important effects on mammalian cells, including these questions it was essential to characterize the gene, or the inhibition of adrenal function (2, 3), chemotaxis of genes, encoding the granulins.t (4), and cytostasis of promyelocytic cell lines in culture (5), and effects on calcium ion flux (6). As part of an METHODS ongoing study of these peptides, we isolated and structurally characterized four members of a family of cysteine-rich Screening of the cDNA Library. To generate the grnA peptides of -6 kDa, which we call granulins A, B, C, and D probe, degenerate oligonucleotide primers [forward primer, (grnA, grnB, grnC, and grnD) (7). No homologous sequences 5'-CGATGTGAAGTG(T/C)GA(T/C)ATGGA-3'; reverse were stored in the databanks, but two partial sequences have primer, 5'-CTGGCATGTGGTT(T/C)TC(A/G)CA(G/A)- been reported that are clearly closely related to the granulins CA-3'] were synthesized (Sheldon Biotechnology Centre, (8). These peptides, called epithelin 1 and 2, were isolated McGill University) and used in the polymerase chain reaction from rat kidneys. Epithelin 1 is mitogenic for in (PCR) with 2 ug of genomic DNA as template (21). The 2 reaction was subjected to 30 cycles of denaturation at 94°C culture, and epithelin 1 and, at lower potency, epithelin for 1 min, annealing at 55°C for 1 min, and elongation at 72°C inhibit the proliferation of certain epithelial cell lines in vitro for 2 min. The amplified product was blunt-ended with T4 (8). We have characterized a granulin-like peptide from rat DNA polymerase and subcloned into the plasmid vector bone marrow (7). The amino terminus of rat bone marrow Bluescript KSII+ (Stratagene), and its identity was con- granulin is identical to the reported partial sequence of renal firmed by double-stranded dideoxynucleotide sequencing epithelin 1, indicating that rat granulins and epithelins are with Sequenase (United States Biochemical). This fragment identical. Consistent with this, human gmnA but not grnB, was labeled with 32P by nick-translation (Boehringer Mann- grnC, or grnD, inhibits proliferation of the epithelial cell line heim) and used to probe a human bone marrow cDNA library A431 in culture (unpublished). in Agtll (Clontech) consisting of 1.51 x 106 independent Growth factors are frequently associated with cells of the clones. Duplicate nitrocellulose filters (Schleicher & Schuell) inflammatory response, and it is generally assumed that this were prehybridized in 5x SSC/5 x Denhardt's solution/0.2% implies important functions for these peptides in tissue repair SDS at 37°C for 5 hr (SSC = 0.15 M sodium chloride/0.015 processes. Examples include platelet-derived growth factor M sodium citrate, pH 7.0; Denhardt's solution = 0.02% (9), which is released into the site of a wound following polyvinylpyrrolidine/0.02% Ficoll/0.02% bovine al- degranulation, transforming growth factor , which is found bumin). Hybridization was in 5 x SSC/2.5 x Denhardt's in platelet granules (10), in (11), and in li- solution/0.2% SDS/50% formamide/10%o polyethylene gly- popolysaccharide-stimulated monocytes (12), and col with 1 x 107 cpm of probe at 37°C for 12 hr. Filters were growth factor, which has been reported in inflammatory washed twice for 45 min in 2x SSC/0.1% SDS at 58°C and ascites (13). Monocytes have recently been shown to gener- ate a heparin-binding growth factor with similarity to epider- Abbreviations: grnA, grnB, grnC, etc., granulins A, B, C, etc.; EGF, epidermal growth factor. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M75161). 1715 Downloaded by guest on September 23, 2021 1716 Biochemistry: Bhandari et al. Proc. Natl. Acad Sci. USA 89 (1992) exposed at -700C with Kodak X-Omat film with an intensi- to probe a human bone marrow cDNA library (Clontech). The fying screen. Sixteen positive clones were obtained from 3 x nucleotide sequence ofthe grnA precursor was then obtained 105 clones screened. The DNA insert from HBM12 was as described in Methods and outlined in Fig. 1B. digested with Kpn I and Sac I, and the resulting fragments The DNA sequence contains an open reading frame of1779 were subcloned into Bluescript KSII+. Nucleotide se- base pairs (bp). The most 5' AUG is probably the start site quences were obtained by double-stranded dideoxy sequenc- because (i) it shows good agreement with the Kozak con- ing using T3, M13, or custom-synthesized 17-mer primers sensus sequence for eukaryotic initiation sites (24) and (ii) we (Sheldon Biotechnology Centre) with Sequenase according have isolated and sequenced a peptide from human granulo- to the manufacturer's instructions. cytes (underlined in Fig. 1C) whose corresponding nucleo- The sequence of HBM12 lacked an initiator methionine tide sequence begins 48 bp 3' to the proposed initiator AUG, codon. The remaining positive clones were analyzed using eliminating AUG codons 3' to this sequence from consider- PCR with Agtll sequence-specific primers in combination with ation. At its 3' end the sequence has the AAUAAA poly(A) a primer, cll2rp, corresponding to nucleotides 45-61 ofclone signal and part of a poly(A) tail. The predicted message, not HBM12. Only two clones, HBM3 and HBM4, contained including a poly(A) tail, is at least 2062 bp, which is in close inserts longerthan HBM12. Their5' sequence was obtainedby agreement with the message length of 2.3 kilobases (kb), dideoxy sequencing ofsingle-stranded templates generated by including a poly(A) tail, determined by Northern blot analysis asymmetric PCR (22) using a forward primer specific for Agtll (see Fig. 4). There is a dichotomy in the 5' untranslated and primer cll2rp as the reverse primer. The 5' sequences of sequences that may be due to the use of alternative splice HBM3 and HBM4 overlapped exactly with HBM12. The sites or alternative exons. Both of these explanations are probability that they were copies from identical transcripts consistent with the observation of two sizes of mRNA was supported by PCR mapping using primer pairs b and d, e transcript in some cell lines (see below). and k, i and k, h and grnAr, grnBfand grnAr, and b and grnFr Deduced Amino Acid Sequence of the Human Granulin (grnAr, grnBf, and grnFr are primers specific for grnA, grnB, Precursor. The deduced sequence ofpreprogranulin (Fig. 1C) and grnF and were not used for sequencing). predicts a of 593 residues, with a probable signal Northern Blot Analysis. Total cellular RNA was isolated peptide (25) extending to residue 17. It contains the 56- from cell lines and rabbit tissues by the acid guanidinium residue grnA sequence, as expected, and also six other thiocyanate/phenol/chloroform extraction method (23). cysteine-rich granulin-like domains, including grnB, grnC, Thirty micrograms (cell lines) or 50 ,ug (rabbit tissues) of and grnD, previously known only from N-terminal sequences RNA was denatured with glyoxal, electrophoresed on a 1.1% (7). The precursor contains two Cys-12 domains, E and F, agarose gel in 10 mM NaH2PO4 (pH 6.8), transferred to nylon and a degenerate granulin domain, G, with 10 cysteines. The membranes (ZetaProbe, Bio-Rad) by capillary blotting with positions ofthe cysteines are highly conserved (Fig. 2), as are 10 mM NaOH, and fixed by baking at 80°C for 2 hr. The certain other residues such as Asp37, His4o, and Pro43, membranes were hybridized at 65C for RNA extracted from numbered according to the ginA sequence (Fig. 2). An eighth cell lines or 60°C for rabbit tissue RNA in 0.5 M NaH2PO4, domain at the amino terminus (paragranulin, Fig. 2) contains pH 6.8/7% SDS/1 mM EDTA for 24 hr with the probe only 6 cysteines that align well with the six amino-terminal described in the legend to Fig. 3. The membranes were cysteines of a granulin domain. We have reexamined peptide washed at hybridization temperature for 30 min in 40 mM extracts of human inflammatory exudate cells for further NaH2PO4, pH 6.8/5% SDS/1 mM EDTA and 30 min in 40 granulin-like peptides and have succeeded in isolating and mM NaH2PO4, pH 6.8/1% SDS/1 mM EDTA. Autoradio- sequencing a peptide corresponding to paragranulin (A.B., grams were obtained by exposing the blots to Kodak X-Omat unpublished). film with intensifying screens at -70°C. There are five potential N- sites (Fig. 1C), Southern Blot Analysis. Zoo-Blots and human Geno-Blots including one at Asn5 of grnC. grnC hydrolysates contain were obtained from Clontech. Hybridization was in 0.5 M amino sugars, and microsequencing (7) gave a blank at NaH2PO4, pH 7.2/7% SDS/1 mM EDTA at 65°C for 20 hr. residue 5 (n = 2), consistent with glycosylation. The inter- Blots were washed twice at 65°C, 30 min each, in 40 mM domain sequences show little homology but contain a Pro-Ala NaH2PO4, pH 7.2/5% SDS/1 mM EDTA and 40 mM dipeptide midway between each domain except F and G. NaH2PO4, pH 7.2/1% SDS/1 mM EDTA. Autoradiography Mono- or dibasic sequences, which are frequent sites of was performed as above. proteolysis in peptide processing (26), flank granulin domains C, D, and E, but not A or B, both ofwhich have been isolated RESULTS as excised peptides. Thus nothing can be reliably inferred Nucleotide Sequence of the Human Granulin Precursor about the post-translational cleavage mechanisms of the cDNA. Extracts ofhuman inflammatory leukocytes contain at granulin precursor. There are no obvious transmembrane least four structurally related granulins (7), and at the outset of sequences beyond the signal peptide (27). these experiments it seemed reasonable that each peptide DNA Blots of the Granulin Precursor. Southern blot anal- could be encoded by a distinct gene. To maximize the spec- ysis of human genomic DNA suggests that only one gene ificity of our probes we decided to use faithfully copied hybridizes with the granulin probe (Fig. 3). Significant inter- fragments of the grnA gene generated by PCR amplification species sequence conservation exists, since DNA blots rather than use small, highly redundant synthetic oligonucle- (Clontech) from human, monkey, rat, mouse, dog, bovine, otide probes ofrelatively low specificity. Two oligonucleotide and rabbit, but not chicken or yeast, hybridized with the primers were synthesized based on probable codon usage and granulin probe (data not shown). The blots probably under- lowest redundancy that corresponded to the amino-terminal estimate species distribution since granulin-like peptides and midportion regions of grnA. grnA was chosen because it have been isolated from teleost hematopoietic tissue is the most abundant granulin in human leukocyte extracts (7), (H. P. J. Bennett, personal communication). and it was the only granulin for which the complete peptide Northern Blot Analysis of Human Granulin Precursor sequence was available (7, 8). Two PCR amplification prod- mRNA. Four myelogenous leukemic cell lines of diverse ucts were obtained from human genomic DNA, both ofwhich lineage, HL-60, U937, K562, KMOE, each expressed a were sequenced. One was unrelated to the granulins or any granulin mRNA of=2.3 kb, as did murine 3T3 fibroblasts. By other sequence stored in the databanks (data not shown). The far the strongest hybridization was detected in epithelial cell other fragment represented part of the ginA gene split by a lines (Fig. 4A). As can be seen in Fig. 4B, two granulin mRNA 79-nucleotide intron (Fig. 1A). This was 32P labeled and used species (2.3 and 2.5 kb) can be distinguished in some of the Downloaded by guest on September 23, 2021 Biochemistry: Bhandari et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1717 A GATGTGAAGTGTGATATGGAGGTGAGCTGCCCAGATGGCTATACCTGCTGCCGTCTACAGTCGGGG 150 160 D V K C D M E V S C P D G Y T C C R L 0 S G CCCACGGGCACCCACCCCCTGGCAAAGAAGCTCCCTGCCCAGAGGACTAACAGGGCAGTG 600 GCCTGGGGCTGCTGCCCTTTTACCCAGgtaccaggtgcggcggtggctoagcacagtgtgcagcag GCCTTGTCCAGCTCGGTCATGTGTCCGGACGCACGGTCCCGGTGCCCTGATGGTTCTACC 660 A L S S S V 11 C P D A R S R C P D G S T ccggccccagtgcccacctgcccttcttcatctgccctagGCTGTGTGTTGTGAAGACCACATCCA 190 200 A V C C E D H I H 720 TGCTGTGAGCTGCCCAGTGGGAAGTATGGCTGCTGCCCMTGCCCMACGCCACCTGCTCCCE P GK Y G C C P * 220AT 210 *220 TCCGATCACCTGCACTGCTGCCCCCMGACACTGTGTGTGACCTGATCCAGAGTMGTC 780 S D H L H C C P Q D T V CD L I Q S KC B 230 240 840 _d h _ CTCTCCMGGAGACGCTACCACGGACCTCCTCACTMGCTGCCTGCGCACACAGTGGGCL S K E N A T T D L L T K L P A H T V G m * 250 260 -______- GATGTGAMTGTGACATGGAGGTGAGCTGCCCAGATGGCTATACCTGCTGCCGTCTACAG 900 a en D V K C D E V S C P D G Y T C C R LO ___-______270 280 U)b fT k O TCGGGGGCCTGGGGCTGCTGCCCTTTTACCCAGGCTGTGTGCTGTGAGGACCACATACAC 960 - G A W G CC P F T 0 A V C C E D H I H c 9 p 290 300 TGCTGTCCCGCGGGGTTTACGTGTGACACGCAGMGGGTACCTGTGMCAGGGGCCCCAC 1020 C6- --P TaKGTCE Q G P H EcoRI KpnI Kpn Sac I EcoRl 310 320 *i CAGGTGCCCTGGATGGAGMGGCCCCAGCTCACCTCAGCCTGCCAGACCCACMGCCTTG 1080 Q V P W M E K A P A HL S L P D P Q A L 330 340 HB1M12 AAGAGAGATGTCCCCTGTGATMTGTCAGCAGCTGTCCCTCCTCCGATACCTGCTGCCM 1140 K R D V P C D N V S S C P S S D T C C Q 350 * 360 HBM4 CTCACGTCTGGGGAGTGGGGCTGCTGTCCMTCCCAGAGGCTGTCTGCTGCTCGGACCAC 1200 L T SGEWGCCPIPEAVC C S D H 370 380 CAGCACTGCTGCCCCCAGCGATACACGTGTGTAGCTGAGGGGCAGTGTCAGCGAGGMGC 1260 Q H C C P Q R Y T C V A E G Q C Q R G S C 390 400 GAGATCGTGGCTGGACTGGAGAAGATGCCTGCCCGCCGCGGTTCCTTATCCCACCCCAGA 1320 5' --- GCGTGGATCCTGAGMCTTCAGGCTCCTGGGCMCGTGCTG E I V A G L E K M P A R R G S L S H P R GTCTTGTGTGCTGGCCCATCACTTTGCAAAGMTTCACCCCACCAG- 410 420 l-CGCAGGCAGACC GACATCGGCTGTGACCAGCACACCAGCTGCCCGGTGGGCGGAACCTGCTGCCCGAGCCAG 1380 5' --- GCGCGGAGTCGGAC- D I G CD a H T S C P V GG T CC P S Q 430 440 ATGTGGACCCTGGTGAGCTGGGTGGCCTTMCAGCAGGGCTGGTGGCTGGAACGCGGTGC 60 GGTGGGAGCTGGGCCTGCTGCCAGTTGCCCCATGCTGTGTGCTGCGAGGATCGCCAGCAC 1440 MS W T L V S W V A L T A G L V A G T R C G G S W A C C Q L P H A V C C E D R Q H 1 450 460 CCAGATGGTCAGTTCTGCCCTGTGGCCTGCTGCCTGGACCCCGGAGGAGCCAGCTACAGC 120 TGCTGCCCGGCTGGCTACACCTGCMCGTGMGGCTCGATCCTGCGAGMGGAAGTGGTC 1500 P D G Q F C P V A C C L D P G G A S Y S C C P A G Y T C N V K A R S C E K E V V 10 20 470 480 TGCTGCCGTCCCCTTCTGGACAMTGGCCCACMCACTGAGCAGGCATCTGGGTGGCCCC 180 TCTGCCCAGCCTGCCACCTTCCTGGCCCGTAGCCCTCACGTGGGTGTGAAGGACGTGGAG 1560 C C R P L L D K W P T T L S R H L G G P S A Q P A T F L A R S P H V G V K D V E 30 40 490 500 TGCCAGGTTGATGCCCACTGCTCTGCCGGCCACTCCTGCATCTTTACCGTCTCAGGGACT 240 TGTGGGGAAGGACACTTCTGCCATGATMCCAGACCTGCTGCCGAGACAACCGACAGGGC 1620 C Q V D A H C S A G H SC I F T V S G T C G E G H F C H D N Q T C C R D N R Q G 50 60 510 520 TCCAGTTGCTGCCCCTTCCCAGAGGCCGTGGCATGCGGGGATGGCCATCACTGCTGCCCA 300 TGGGCCTGCTGTCCCTACGCCCAGGGCGTCTGTTGTGCTGATCGGCGCCACTGCTGTCCT 1680 S S CC P F P E A V A C G D G H H C C P W A CC P Y A Q G V C C A D R R H C C P 70 80 530 540 CGGGGCTTCCACTGCAGTGCAGACGGGCGATCCTGCTTCCAAAGATCAGGTMCMCTCC 360 GCTGGCTTCCGCTGCGCACGCAGGGGTACCMGTGTTTGCGCAGGGAGGCCCCGCGCTGG 1740 R G F H C S A D G R S C F Q R S G N N S A G F R C A R R G TK C L R R E A P R W 90 100 * 550 560 GTGGGTGCCATCCAGTGCCCTGATAGTCAGTTCGMTGCCCGGACTTCTCCACGTGCTGT 420 GACGCCCCTTTGAGGGACCCAGCCTTGAGACAGCTGCTGTGAGGGACAGTACTGAAGACT 1800 V G A I C C P D S 0 F E C P D F ST C C D A P L R D P A L R 0 L L 110 120 570 GTTATGGTCGATGGCTCCTGGGGGTGCTGCCCCATGCCCCAGGCTTCCTGCTGTGMAGAC 480 _.CTGCAGCCCTCGGGACCCCACTCGGAGGGTGCCCTCTGCTCAGGCCTCCCTAGCACCTCC_..__vv _ _uzul I_ ___,I v_I_,___I___,~v ___, I_____1 860 __ V M V D G S W G C C P M P a A S C C E D CCCTMCCAAATTCTCCCTGGACCCCATTCTGAGCTCCCCATCACCATGGGAGGTGGGGC 1920 130 140 CTCMTCTAAGGCCCTTCCCTGTCAGAAGGGGGTTGAGGCAAAAGCCCATTACAAGCTGC 198(1 AGGGTGCACTGCTGTCCGCACGGTGCCTTCTGCGACCTGGTTCACACCCGCTGCATCACA 540 CATCCCCTCCCCGTTTCAGTGGACCCT1TGGCCAGGTGCTTTTCCCTATCCACAGGGGTG 2040 R V H C C P H G A F C D L V H T R C I T TTTGTGTGTTGGGTGTGCTTTc ;~~TTTGTC ACTTTCTTAA 2099 FIG. 1. (A) Sequence of the probe used to screen a human bone marrow cDNA library in Agt11 (Clontech). The probe was generated by PCR of human DNA. Underlined sequences correspond to forward and reverse primers, and the nucleotides in lowercase letters represent an intron in the coding region of grnA. (B) Sequencing strategy. Arrows indicate the direction of sequencing and overlap and are explained in the text. (C) Complete nucleotide sequence and deduced polypeptide sequence of granulin. The nucleotides are numbered from the initiator codon (ATG), and the amino acids are numbered from the probable signal peptide cleavage site. Underlined sequences correspond to sequences previously determined by gas-phase microsequencing of purified granulins (ref. 1; unpublished). Possible N-glycosylation sites are indicated by an asterisk (*), and the stop codon is shown by #. The boxed nucleotides correspond to a poly(A) signal. Clone HBM12 starts at nucleotide 6. The sequences of clones HBM3 and HBM4 differ in the 5' untranslated region, as indicated by the bifurcation in the nucleotide sequence. cell lines. Determination of the origin and significance of the obtained with rabbit kidney RNA after 4 days, and much two mRNA species requires further investigation. In total weaker hybridization was obtained with spleen RNA (Fig. RNA extracts from tissues, very strong hybridization was 4C). After prolonged exposure (25 days) hybridization was

GRANULIN A DVK DMEVS- CPDGYTCCRLQSG AW- CCPFTQAVCC E lH I HCCP AS3FT DTQKGT E GRANULIN B VM PDAR S|R CP DGS TCC E LP SG K jGj- CCPMPNAFCC H L HCCP QDTV DL QSK L GRANULIN C P D N V S|S|- CP SSD TCCQL T SG E W G- CCOPIEAVCC HC HOCP QRYT VAEGQ- 0 GRANULIN D I G DQHT[S- CPVGGTCCPSQG[ S WA- COOLMHH CC RO HCCP AGjYT NVKA RS E GRANULIN E DfV E G EGHF- CHHD NQ TCC RDNRQGW A- CCPYAQGVCC RRHCCP AIGIFR ARRG TK L GRANULIN F A Q P DSQFE CPD FS TCCVMVVGS W OCPM{QAC R V HCCP HIGIAF DLV HTR

GRANULIN G GG P|C Q V D A H - CSAGHS FTV SG TS S- C CPFPEAV GH HCCP R G FH SADGRS F PARAGRANULIN TRM P DGQF- VAA-- LDPGL AS YS CORP LLD

FIG. 2. Comparison of the granulin-like domains. Residues occurring four or more times are boxed, and dashes have been introduced to align the cysteines. The domain boundaries were determined by gas-phase microsequencing (ref. 1; unpublished data) and by comparison with the sequence of grnA. The domains are located at amino acids 264-319 (grnA), 169-244 (grnB), 347-400 (grnC), 425-479 (grnD), 501-556 (gmE), 106-162 (grnF), 41-96 (gmG), and 1-27 (paragranulin). The order of domains is paragranulin, G, F, B, A, C, D, E. They are not alphabetically aligned because several domains were isolated and named as discrete before their common origin was known. Downloaded by guest on September 23, 2021 1718 Biochemistry: Bhandari et al. Proc. Natl. Acad Sci. USA 89 (1992) Multiple sequential repeats of cysteine-rich domains is a _ Fr- or recurrent structural motifamong regulatory proteins (28-31). en co .E U M X m IX Typically, one exon delineates one cysteine-rich domain kb (31-33) but, in contrast, the PCR-amplified fragment of grnA 23.5 - is bisected by an intron (Fig. 1A), and preliminary results 9.4 qpww show that introns bisect domains G, F, B, C, and D (details 6.7 will be published elsewhere). In each case the cysteines align 4.4 with an approximate mirror-image symmetry around the bisecting intron. The iterative nature of progranulin draws unavoidable comparisons with the EGF precursor (28, 29). 2.3 The progranulin and EGF precursor (34) mRNAs are very 2.0 abundant in the kidney relative to other tissues, and, biolog- ically, EGF and granulin/epithelins have similar actions on epithelial cells but not on fibroblasts (8), although they probably act through different receptors (8). It is presently unclear whether these similarities are biologically significant FIG. 3. Southern blot analysis of human DNA, digested with Bg1 or merely coincidental. Unlike the EGF precursor, progran- II, Pst I, BamHI, HindIII, and EcoRI, 5 ,ug per lane (Clontech), and ulin has no transmembrane segment or cytoplasmic domain probed with a 32P nick-translated 1890-bp EcoRI/Sac I fragment (28, 29), implying different post-translational pathways and from clone HBM12. receptor presentation strategies for the two proteins. Several lines of evidence point to a transit of the granulin precursor detected using total RNA from liver, heart, lung, thymus, and through the rough endoplasmic reticulum/Golgi apparatus brain, but not in skeletal muscle (data not shown). compartment. (i) There is the presence of a probable signal DISCUSSION sequence (25). (ii) grnC is glycosylated and glycosylation occurs exclusively in the rough endoplasmic reticulum and The presence of all four known granulins and three granulin- Golgi apparatus. (iii) Granulins are disulfide bridged (7, 8), like sequences in a common precursor was unexpected. implying exposure to the enzyme protein disulfide isomerase, which is located in the rough endoplasmic reticulum (35). (iv) A In human granulocyte extracts, the granulins copurified with C!) a granular fraction free of cytoplasmic marker peptides (7). 0 LUJ (° II- N ) Whether the peptides enter a constitutively secreted, or CDs Lt I4 CIO .s) regulated, pathway (36) awaits further studies. The process- I m y y < < CY) ing of growth factors and related polypeptides can follow several pathways. Some, such as transforming growth factor (3, are cleaved from their precursors at canonical dibasic sites 2.4kb *W, 0 (37); others, such as EGF, are cleaved at monobasic sites (28, 9eis 29); and many, such as transforming growth factor a (38), a (39), and interleukin 1,8 (40), are cleaved from their precursors at small nonbasic residues. Basic and nonbasic potential cleavage sites are found in the B granulin precursor. In one case of chronic myelogenous Cl) leukemia in blast crisis we found that a granulin-D-like LU 0)2 molecule was the dominant peptide (unpublished), whereas

lq 10 ~e in normal peripheral leukocytes gmnA is the major product <

2.4kb .. , translational cleavage in different cells. The abundant expression of granulin mRNA in epithelial cell lines is particularly interesting, since epithelial cells, including A431 (8), respond to, and presumably have recep- tors for, epithelin 1 (and ginA; unpublished). Many solid tumors are epithelial in origin; thus the propensity of trans- C H L Sp K BM formed epithelial cells to express granulin mRNA, and re- spond to the mature peptide, may have pathophysiological consequences. We initially found granulins in inflammatory cells and bone marrow (7), so the presence ofgranulin mRNA 2.4kb in HL-60 and U937 cells was expected. The expression ofthe granulin gene in two proerythroid cells, K562 and KMOE, was unanticipated. Proerythroid cells and their progeny are unlikely to be involved in regulating epithelial proliferation, suggesting other possible functions for the granulins. The FIG. 4. Northern blot analysis. (A) Granulin precursor mRNA in strong expression of granulin mRNA in the kidney conclu- the following cell lines: HL-60 (promyelocytic leukemia), U937 sively proves that the granulin/epithelin peptides are intrin- (histiocytic leukemia), K562 and KMOE (proerythroid leukemias), sically renal (7, 8) and may imply functions unrelated to A431 (epidermoid carcinoma), A549 (lung epithelial carcinoma), epithelial cell mitogenesis, as has been suggested for renal SKMES-I (lung squamous carcinoma), CHO-KI (Chinese hamster ovaries), and murine BALB/c 3T3 fibroblasts, exposed for 10 days. EGF (34). Thus granulins may have multiple biological ac- (B) RNA from A431, A549, and SKMES-I, exposed for 20 hr. (C) tivities; moreover, distinct activities may be associated with Distribution ofgranulin mRNA in rabbit tissues: heart (H), liver (L), different domains, as already proposed for epithelins 1 and spleen (Sp), kidney (K), and bone marrow (BM), exposed for 4 days. 2 (8). Downloaded by guest on September 23, 2021 Biochemistry: Bhandari et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1719 The physiological functions of granulin/epithelins remain Francis, N., Wahl, L. M., Roberts, A. B. & Sporn, M. B. largely speculative. Cumulatively, however, the evidence (1987) Proc. Natl. Acad. Sci. USA 84, 5788-5792. is 16. Reibman, J., Meixler, S., Lee, T. C., Gold, L. I., Cronstein, suggests a role of some significance. The granulin gene B. N., Haines, K. A., Kolasinski, S. L. & Weissmann, G. widely expressed in cells of diverse lineage; some granulins/ (1991) Proc. Natl. Acad. Sci. USA 88, 6805-6809. epithelins have potentially important effects on epithelial cell 17. Ignotz, R. A. & Massague, J. (1986) J. Biol. Chem. 261, proliferation (8), and, as noted previously, there is a high 4337-4345. degree of sequence conservation (7), corroborated here by 18. Bauer, E. A., Cooper, T. W., Huang, J. S., Altman, J. & interspecies DNA hybridization. Our initial studies were Deuel, T. F. (1985) Proc. Natl. Acad. Sci. USA 82, 4132-4136. based on the premise that granulins were involved in the 19. Postlethwaite, A. E., Keski-Oja, J., Moses, H. L. & Kang, the results of Northern A. H. (1987) J. Exp. Med. 165, 251-256. inflammatory response; however, 20. Sporn, M. B. & Roberts, A. B. (1986) J. Clin. Invest. 78, blots (Fig. 4) show that the granulin gene is expressed in a 329-332. much wider context. The availability of a cloned granulin 21. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, precursor cDNA will allow us to study where and under what R., Horn, G. T., Mullis, K. B. & Erlich, H. A. (1988) Science circumstances the granulin gene is expressed and what fac- 239, 487-491. tors regulate that expression. 22. Gyllensten, U. B. & Erlich, H. A. (1988) Proc. Natl. Acad. Sci. USA 85, 7652-7656. We acknowledge the energetic support of Dr. Samuel Solomon, 23. Chomczynski, P. & Sacchi, N. (1987) Anal. 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