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Isolation and Sequence of the Granulin Precursor Cdna from Human Bone

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Isolation and Sequence of the Granulin Precursor Cdna from Human Bone 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 growth factor (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 fibroblasts 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. gene 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 inflammation 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 genes 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 monocytes (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 keratinocytes 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 serum 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 neutrophils (11), and in li- solution/0.2% SDS/50% formamide/10%o polyethylene gly- popolysaccharide-stimulated monocytes (12), and fibroblast 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 protein 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).
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