Eur. J. Biochem. 189,287-293 (1990) 0 FEBS 1990
Type 5 acid phosphatase Sequence, expression and chromosomal localization of a differentiation-associated protein of the human macrophage Deirdre K. LORD', Nicholas C. P. CROSS', Maria A. BEVILACQUA', Susan H. RIDER3, Patricia A. GORMAN3, Ann V. GROVES3, Donald W. MOSS', Denise SHEER3, and Timothy M. COX' Department of Haemdtology, The Royal Postgraduate Medical School, London, England Department of Chemical Pathology, The Royal Postgraduate Medical School, London, England Cytogenics Laboratory, Imperial Cancer Research Fund Laboratories, London, England (Received October 4, 1989) - EJB 89 1203
The purple acid phosphatases and uteroferrin belong to a diverse multifunctional class of binuclear ironcontaining proteins that includes haemerythrin and ribonucleotide reductase. In the pig, uteroferrin has been implicated in the delivery of iron to the foetus, but the role of the related human type 5 acid phosphatase that is principally found in resident tissue macrophages is not yet clear. To define further the function of this metalloenzyme, we have isolated and sequenced a cDNA clone for type 5 acid phosphatase and investigated expression of its gene in human tissues. The phosphatase clone contains an open reading frame of 975 bp and encodes a protein of 325 amino acids, including a signal peptide of 19 residues and two potential sites for N-glycosylation. The type 5 acid phosphatase gene mapped to the short arm of human chromosome 19 and was found to have a restriction fragment length polymorphism on digestion with XbaI. Expression of phosphatase mRNA was restricted to mononuclear phagocytes and the enzyme was induced > 20-fold on transformation of normal human monocytes to macrophages by culture in serum-supplemented medium. Type 5 acid phosphatase thus represents a tightly regulated system for the study of molecular events in the differentiation programme of the normal macrophage. Type 5, tartrate-resistant, purple acid phosphatase is a basic, iron-binding protein with high activity towards phosphoproteins, ATP, and 4-nitrophenyl phosphate but with little action on aliphatic phosphates [I, 21. The isoenzyme is found in human alveolar macrophages [3] and osteoclasts [4] and is pathologically increased in Gaucher's cells [5] and in the hairy cells of leukaemic reticuloendotheliosis [6], now known as hairy cell leukaemia. It is thus expressed principally in cells of the human mononuclear macrophage system. Recently the protein has been purified from human and bovine spleen, where it is associated with a particulate fraction [7, 81. lmmunochemical and enzymatic studies [9, 101 as well as protein sequence analysis [I 11 have shown that the splenic enzyme is homologous to uteroferrin, a purple iron-binding protein abundant in the uterine secretions of pregnant sows. Physiological studies in vivo have implicated uteroferrin in the delivery of maternal iron to the developing piglet [12, 131 but the possibility of a similar function for the human tartrateresistant acid phosphatase has not been investigated. However, ultrastructural studies [14] indicate association of the bovine protein with ferritin and haemosiderin aggregates in splenic macrophages, suggesting that it may participate in the Correspondence to T. M . Cox, Dept. Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, England Note. The novel nucleotide sequence data published here has been deposited with EMBL sequence data bank and is available under accession number X14618. Enzymes. Type 5 acidphospkatase (EC 3.1.3.2); type I1 restriction endonucleases (EC 3.1.21.4).
retrieval of iron released by erythrophagocytosis. In addition, there is recent evidence that the enzyme is essential for bone resorption by human osteoclasts [15]. To investigate the function and expression of the type 5 acid phosphatase isoenzyme in human tissues, we have used a novel procedure to isolate a cDNA probe from human spleen and have utilized this to characterize full-length clones from a human cDNA library. We have further utilized the full-length cDNA to monitor expression of type 5 acid phosphatase in human tissues and to determine the chromosomal localization of its gene. MATERIALS AND METHODS cDNA amplification
Poly-(A)-rich RNA, purified from Gaucher's spleen, was reverse-transcribed using an oligo(dT) primer [16]. The RNA was hydrolysed by addition of NaOH to 66mM and incubating at 65 "C for 1 h. After neutralization with HCI in the presence of 0.2M Tris, the single-stranded cDNA was ethanolprecipitated and resuspended in 10 mM Tris/HCl, 0.1 mM EDTA pH 8.0. Published amino acid sequences for porcine uteroferrin and acid phosphatase from bovine spleen [ l l ] were used to derive oligonucleotide primers for use in the polymerase chain reaction to amplify human phosphatase sequences from the single-stranded cDNA. Oligonucleotides were synthesized to provide all possible sequences coding for two regions conserved between porcine uteroferrin and bovine spleen phos-
288 phatase. Sense oligonucleotides were made to FQETFED and antisense to GHYPVW. Each pool of oligonucleotide primers was highly degenerate and contained 256 individual sequences. The polymerase chain reaction was carried out using 100 ng cDNA and 2.5 U Taq polymerase (Perkin Elmer Cetus), as recommended by the manufacturer, except that the primer concentration was increased to 10 pM. The reaction was carried out in a home-made thermocycling apparatus 1171 under the following conditions: 92"C, 2 min; 45"C, 2 min; 72"C, 2 min. After 30 cycles, a final extension of 10 min at 72°C was made and an aliquot of the reaction mixture was analysed on a 2% agarose minigel. Amplified DNA was gelpurified by the freeze-squeeze technique [18], treated with polynucleotide kinase [16]and ligated into pEMBL19 cut with Smal and dephosphorylated.
trifugation through discontinuous plasma/Percoll gradients, as described by Savill et al. [23]. The upper layer of mononuclear cells was removed from the underlying polymorphonuclear cells, washed and resuspended at 4 x lo6 cells/ml in Iscove's modified Dulbecco's medium. 1ml was added to each well of 24-well tissue culture plates. After a 1 h incubation at 37°C and 6% C 0 2 , the wells were washed and adherent monocytes cultured for three days in Iscove's medium with 10% autologous serum. Assay of type 5 human acidphosphatase Type 5 tartrate-resistant acid phosphatase activity was assayed using the immunoenzymatic method described by Whitaker et a1 1241with 4-nitrophenyl phosphate as substrate. Chromosomal mapping
cDNA library screening A 1" g t l l cDNA library (Clontech) made from human placenta was used for screening. Plaque and colony hybridization was carried out according to Maniatis et al. [16]. CDN A probes cDNA probes were labelled with [ W ~ ~ P I ~ C using T P the multiprime DNA-labelling system (Amersham). D N A sequencing Restriction fragments were subcloned into pEMBL 18 or 19 and both strands were sequenced by the chain-terminator method [I91 using the M13 universal primer (New England Biolabs). The cloning and sequencing strategy is shown in Fig. 1. Southern blot analysis DNA was extracted from peripheral blood samples by urea/SDS lysis [20], digested, electrophoresed in an 0.8% agarose gel and then transferred to Hybond N membrane (Amersham) as described by Southern [21]. Hybridization with the phosphatase probe was carried out at 65°C in 6 x NaCl/Cit (NaCl/Cit: 0.15 M NaC1/15 mM trisodium citrate, pH 7.5) 0.1% SDS, 5 x Denhardt's, 100 pg/ml salmon sperm DNA for 16 h. Filters were washed in 1 x NaCI/Cit 0.1 % SDS and 0.2 x NaCl/Cit 0.1 % SDS at 65 "C for 1 h. Northern blot analysis Total RNA was prepared from human tissues by the guanidinium isothiocyanate method [22] and quantified by ultraviolet spectroscopy [16]. 10-pg samples were electrophoresed on 1.5% formaldehyde/agarose gels, transferred to Hybond N membranes (Amersham) and hybridized at 42 "C in 5 x Denhardt's solution, 6 x NaCl/Cit, 0.5% SDS, 50% formamide, 100 pg/ml salmon sperm DNA for 16 h. The filters were washed once in 10 x Denhardt's, 6 x NaCl/Cit, 0.1 % SDS and then twice in 3 x NaCl/Cit, 0.1% SDS at 65°C for 30 min.
Cells and D N A preparation. Cell lines were maintained in RPMI 1640 medium with 10% foetal calf serum at 37°C under an atmosphere of 95% 02/5% C 0 2 . To retain human chromosome 17 or X, where appropriate, media were supplemented with 100 pM hypoxanthine, 10 pM methotrexate and 10 pM thymidine. All 42 hybrid cell lines have been previously described (references available on request from DKL) and their chromosome content was defined by enzymatic and karyotypic analysis. Assays for human enzymes were carried out using standard methods [25] and the karyotypes were determined by a combination of cell staining and quinacrine banding [26, 271. Approximately 5 x lo7 fresh cells were used for extraction of high-molecular-mass genomic DNA [28]. 30 pg hybrid DNA, parental rodent DNA and 15 pg human DNA (obtained from fresh lymphocytes) were digested with BamHI (BRL) and electrophoresed for Southern blotting as described above. A 1.4-kb EcoRI fragment, representing a full-length cDNA was used to probe the phosphatase gene after hybridization to high stringency with 0.1 x NaCl/Cit, 0.1% SDS at 65 "C. The washed filters were exposed to XAR5 film (Kodak) with intensifying screens (Dupont) at - 70°C for up to seven days. In situ hybridization Human lymphocytes were cultured with phytohaemagglutinin for 72 h at 37°C and bromodeoxyuridine was added to give a final concentration of 200 pg/ml [29]. After 16 h, the cells were washed and further incubated in thymidinesupplemented medium for 6 h. Cell harvesting and all further procedures were carried out in subdued lighting. In situ hybridization was based on the method of Harper and Saunders [30] using acid phosphatase cDNA labelled with [3H]deoxyribonucleotidesto a specific activity of 3 x 10' cpm/ pg. The hybridization was carried out at 37°C with probe concentrations of 0.02 pg/ml and 0.1 pg/ml. Slides were washed in 2 x NaCl/Cit at 39"C, dehydrated and coated with Ilford K5 emulsion. Slides were developed after 6 - 10 days and G-banded by a procedure based on the method of Wolff and Perry [31] RESULTS AND DISCUSSION
Preparation of monocyte-derived macrophages
Cloning of tartrate-resistant phosphatase
Mononuclear cells were isolated from citrated peripheral blood by a combination of dextran sedimentation and cen-
Human acid phosphatase was cloned by a novel procedure that utilizes the polymerase chain reaction to amplify cDNA
289
c
2
wl
a W
4
h
-
-+--c---
Fig 1 . Restriction map and sequencing strategy for the tartrate-resistant acidphosphatase cDNA clone HP3. The position of restriction sites used in sequencing are indicated. The solid box represents the coding region, the lines designate the 5’- and 3’-untranslated regions. Arrows show the direction and the extent of each sequence determination
using pools of oligonucleotide primers designed on the basis of the protein sequence [32]. Specifically, we selected regions shared between bovine acid phosphatase and porcine uteroferrin and we synthesized mixed oligonucleotides with a high degree of degeneracy. In uteroferrin the distance between these sequences is 115 amino acid residues, but attempts to amplify the human gene from genomic DNA were unsuccessful, presumably because of the presence of one or more introns in this region. Thus single-stranded cDNA was used as the template for the reaction. The RNA used for reverse transcription was obtained from the spleen of a patient with Gaucher’s disease, a disorder in which expression of type 5 acid phosphatase is enhanced. Analysis of the reaction products after amplification showed a major fragment of 330 bp, i.e. approximately the predicted size. The 330-bp DNA was cloned to yield the plasmid pPCR330, the insert of which was sequenced. The derived amino acid sequence of this clone was found to be highly homologous to the corresponding sequence of porcine uteroferrin and bovine spleen acid phosphatase. pPCR330 was used to screen 1.25 x l o 5 plaques of a human placental cDNA library in A g t l l . Three putative clones were plaque-purified, digested with EcoRI and found to contain 1.3- 1.4-kb inserts. The insert from the largest of these (A HP3) was subcloned and sequenced following the strategy shown in Fig. 1. Fig. 2 shows the 1359-nucleotide sequence and deduced amino acid sequence of the human type 5 acid phosphatase. Acid phosphatase cDNA comprises a 35-bp 5’-untranslated region followed by one of two possible in-frame initiator methionines both of which lie downstream of an in-frame stop codon at nucleotide 12-14. The open reading frame is 975 bp and encodes 325 amino acids that correspond to a polypeptide of molecular mass 36571 Da. The 3’-non-coding region is 346-bp long with a potential polyadenylation signal 19 bp upstream of the poly(A) tract. The 5‘ end of one of the smaller cDNA insert HP1 was sequenced and found to be identical to HP3 but started slightly downstream at nucleotide 186. The nucleotide sequence of full-length acid phosphatase is identical to that of pPCR330, except for the replacement of C at positions 424 and 519 by G. This discrepancy between the spleen and placental sequences may represent allelic variations or reflect replication errors by Tuq polymerase during the polymerase chain reaction. The NH,-terminal region of acid phosphatase contains many hydrophobic residues that are characteristic of a signal peptide. The assignment of the cleavage site between resi-
dues -1 and + l is based on Von Heijne’s (-3,-I) rule [33]. However, this remains tentative until the NH,-terminal residue of the mature protein is known. The sequence predicts two possible glycosylation sites (Asn-Val-Ser) for asparaginelinked oligosaccharides at residues 97 and 128. Type 5 human acid phosphatase, bovine spleen phosphatase and uteroferrin are homologous proteins with similar physical and enzymatic properties. Moreover, comparative partial sequence studies have confirmed that there is greater than 75% conservation at the amino acid level between bovine spleen phosphatase and porcine uteroferrin [l 11. In common with the type 5 phosphatases, uteroferrin possesses acid phosphatase activity but it appears to be a predominantly secreted protein, whereas the type 5 acid phosphatase is found within cells and has been localized to membrane-bound organelles. The mannose 6-phosphate residue of uteroferrin has been shown in biosynthetic labelling experiments to be masked, and this may explain its extrusion by endometrial cells in culture [34]. Comparison of the predicted amino acid sequence of the human type 5 phosphatase with the human prostatic [35] and lysosomal [36] acid phosphatases revealed no sequence similarity and additional searches have failed to show any significant resemblance to other known lysosomal enzymes. Computer-assisted analysis did not reveal an obvious hydrophobic membrane-spanning domain. Since completing the sequence of our cDNA clones, Ketcham et al. have published a sequence of type 5 acid phosphatase from human placenta [37]. There are four discrepancies between the sequence reported here and that published by Ketcham et al. Two residues (Lysl60 and Leul61) were found in this study, in the phosphatase clone from human spleen and in the human placental sequence, that are not present in Ketcham’s sequence, which predicts a protein that is two amino acids shorter. However, because our sequence was found in cDNA from two independent sources, we believe it to be correct. We also found Ala and Arg at positions 26 and 27 of the protein where Gly and Pro were reported by Ketcham et al. The explanation for this is unclear but these discrepancies arise in a region found to be subject to sequence compression that we resolved by the use of ITP in the sequencing reactions. Moreover, the sequence contains a SmaI site that was used in cloning (see Fig. 1). Previous studies have indicated that iron, which is essential for the enzymatic activity of uteroferrin and spleen acid phosphatase, is present in these proteins as an anti-ferromagnetically coupled binuclear p o x 0 core [38]. The mixed valence ferric-ferrous couple of the pink, reduced form of these pro-
290 AGAGCCTCCGGTGACTGGCCTGTGTCTCCCCCTGG --19 -10 Met Asp Met Trp Thr Ala Leu Leu Ile Leu Gln Ala Leu Leu Leu ATG GAC ATG TGG ACG GCG CTG CTC ATC CTG CAA GCC TTG TTG CTA
35
I
2
3
L
5
6
7
8
I
I
I
I
I
I
I
I
80
10 -.
-1. +1-
Pro Ser Leu Ala Asp Gly Ala Thr Pro Ala Leu Arg Phe Val Ala CCC TCC CTG GCT GAT GGT GCC ACC CCT GCC CTG CGC TTT GTA GCC
125
20
Val Gly Asp Trp Gly Gly Val Pro Asn Ala Pro Phe H i s Thr Ala GTG GGT GAC TGG GGA GGG GTC CCC AAT GCC CCA TTC CAC ACG GCC 30
170
40
Arg Glu Met Ala Asn Ala Lys Glu Ile Ala Arg Thr Val Gln Ile CGG GAA ATG GCC AAT GCC AAG GAG ATC GCT CGG ACT GTG CAG ATC
- 285
215
50
Leu Gly Ala Asp Phe Ile Leu Ser Leu Gly Asp Asn Phe Tyr Phe CTG GGT GCA GAC TTC ATC CTG TCT CTA GGG GAC AAT TTT TAC TTC 60
2 60
70
Thr Gly Val Gln Asp Ile Asn Asp Lys Arg Phe Gln Glu Thr Phe ACT GGT GTG CAA GAC ATC AAT GAC AAG AGG TTC CAG GAG ACC TTT
305
-185
80
Glu Asp Val Phe Ser Asp Arg Ser Leu Arg Lys Val Pro Trp Tyr GAG GAC GTA TTC TCT GAC CGC TCC CTT CGC AAA GTG CCC TGG TAC ~
90
350
100
Val Leu Ala Gly Asn H i s Asp H i s Leu Gly Asn Val Ser Ala Gln GTG CTA GCC GGA AAC CAT GAC CAC CTT GGC AAT GTC TCT GCC CAG
- 1.5 kb
395
110
Ile Ala Tyr Ser Lys Ile Ser Lys Arg Trp Asn Phe Pro Ser Pro ATT GCA TAC TCT AAG ATC TCC AAG CGC TGG AAC TTC CCC AGC CCT 120 130 Phe Tyr Arg Leu H i s Phe Lys Ile Pro Gln Thr Asn Val Ser Val TTC TAC CGC CTG CAC TTC AAG ATC CCA CAG ACC AAT GTG TCT GTG
440 485
140
Ala Ile Phe Met Leu Asp Thr Val Thr Leu Cys Gly Asn Ser Asp GCC ATT TTT ATG CTG GAC ACA GTG ACA CTA TGT GGC AAC TCA GAT 150
530
160
Asp Phe Leu Ser Gln Gln Pro Glu Arg P r o Arg Asp Val Lys Leu GAC TTC CTC AGC CAG CAG CCT GAG AGG CCC CGA GAC GTG AAG CTG
515
170
Ala Arg Thr Gln Leu Ser Trp Leu i y s Lys Gln Leu Ala Ala Ala GCC CGC ACA CAG CTG TCC TGG CTC AAG AAA CAG CTG GCG GCG GCC 180
620
190
Arg Glu Asp Tyr Val Leu Val Ala Gly AGG GAG GAC TAC GTG CTG GTG GCT GGC 200 Ile Ala Glu H i s Gly Pro Thr H i s Cys ATA GCC GAG CAC GGG CCT ACC CAC TGC 210 Pro Leu Leu Ala Thr Tyr Gly Val Thr CCA CTG CTG GCC ACA TAC GGG GTC ACT
H i s Tyr Pro Val Trp Ser CAC TAC CCC GTG TGG TCC
665
Leu Val Lys Gln Leu Arg CTG GTC AAG CAG CTA CGG
710
220
Ala Tyr Leu Cys Gly H i s GCC TAC CTG TGC GGC CAC
755
ASP H i s Asn Leu Gln Tyr Leu Gln Asp Glu Asn Gly Val Gly Tyr GAT CAC RAT CTG CAG TAC CTG CAA GAT GAG AAT GGC GTG GGC TAC
800
?in --”
240
250
Val Leu Ser Gly Ala Gly Asn Phe Met Asp Pro Ser Lys Arg His GTG CTG AGT GGG GCT GGG AAT TTC ATG GAC CCC TCA AAG CGG CAC
845
260
Gln Arg Lys Val Pro Asn Gly Tyr Leu Arg Phe H i s Tyr Gly Thr CAG CGC AAG GTC CCC AAC GGC TAT CTG CGC TTC CAC TAT GGG ACT
890
280
270
Glu Asp Ser Leu Gly Gly Phe Ala Tyr Val Glu Ile Ser Ser Lys GAA GAC TCA CTG GGT GGC TTT GCC TAT GTG GAG ATC AGC TCC AAA
935
290 ~~
~
Glu Met Thr Val Thr Tyr Ile Glu Ala Ser Gly Lys Ser Leu Phe GAG ATG ACT GTC ACT TAC ATC GAG GCC TCG GGC AAG TCC CTC TTT 980 300 Lys Thr Arg Leu Pro Arg Arg Ala Arg Pro * AAG ACC AGG CTG CCG AGG CGA GCC AGG CCC TGA ACTCCCATGACTGCC 1028 CAGCTCTGAGGCCCGATCTCCACTGTTGGGTGGGTGGCCTGCCGGGACCCTGCTCACAGG 1088 CAGGCTTTTCCTCCAACCTGTGGCGCTGCAGCAGGGCAGGAAGGGGAAACACAGCTGATG 1148 RACTGTGGTGCCACATGACCCTTGTGGCACAGATGCCCACGTATGTGAAACACACATGGA 1208 CATGTGTCCCAGCCACAGTGTTATGCTCTGTCGCTGGCTGGCTCACCTTTGCTGAGTTCCGGGG 1268 TGCAATGGGGGAGGGAGGGAGGGAAAGCTTCCTTCCTCCT~TCAAGCATCTTTCTGTTACTG 1328 ATGTTC-GAATAGTTGCCAAGGCTG
1359
Fig 2. Nucleotide and deduced amino acid sequence of human tartrateresistant acid phosphatase cDNA. The signal peptidase cleavage site follows amino acid - 1, and the + 1 amino acid is assigned to the first residue of the mature protein. The two possible N-linked glycosylation sites (Asn-Val-Ser) are at residues 97-99 and 128- 130. The termination codon is indicated by an asterisk and the putative polyadenylation signal in the 3’-untranslated region is underlined
teins generates an intense EPR signal at g’ = 1.74 [39, 401. An almost identical signal has been detected in other ironcontaining proteins, e.g. ribonucleotide reductase [41], and this feature appears to be diagnostic of a unique class of metalloproteins. Although the binuclear iron-containing proteins are characterized by specific EPR signals, with the exception of their unique iron centre, they show marked divergence in their amino acid sequences, which probably reflects their different functions. On the other hand, the purple acid phosphatases and uteroferrin show remarkable sequence conservation, which suggests a common functional role. There is evidence that histidine and tyrosine residues are involved in the binding of the two iron atoms [2, 401 (and
Fig. 3. Northern blot analysis of 10-pg aliquots of RNA from human tissues and cells using full-length acid phosphatase cDNA as a probe. Total RNA was isolated from Gaucher’s spleen (lane l), normal spleen (lane 2), bone marrow (lane 3), reticulocytes (lane 4), lymphocytes (lane 5), liver (lane 6), monocytes (lane 7) and monocyte-derived macrophages (lane 8). A 1.5-kb mRNA for acid phosphatase is seen in samples from Gaucher’s disease spleen and macrophages. Markers at the right of the figure show the position of 28s and 18s rRNA. The exact size of the acid phosphatase transcript was determined using RNA calibration standards (BRL)
references therein), and a search for amino acid repeats in human acid phosphatase revealed a His/Tyr cluster at positions 86-94 and an almost exact mirror-image sequence at positions 221 - 227, which may thus constitute iron-binding domains. In ribonucleotide reductase, the active centre tyrosine at position 122 is found in conjunction with a highlyconserved histidine residue at position 118 [42]; a configuration that is conserved between uteroferrin, bovine spleen phosphatase and human type 5 acid phosphatase at positions 223 (His) and 227 (Tyr). This region appears to be the most plausible candidate for the iron-binding site, but the occurrence of two similar domains in human acid phosphatase raises the possibility that four rather than two iron atoms may be bound for each polypeptide, as suggested by analysis of the enzyme isolated from human osteoclasts [43]. Expression of type 5 acidphosphatase in human tissues RNA from a range of human tissues and cells was probed with the labelled cDNA insert from pHP3. RNA with a length of 1.5 kb was found to be expressed in large amounts in Gaucher’s spleen and macrophages (see Fig. 3). A barely detectable signal was seen in resting monocytes, liver and lymphocytes and none was detected in total RNA from control
29 1 Table 1. Type 5 acidphosphatase in human monocytes andmacrophages Type 5 acid phosphatase activity was determined immunoenzymatically as described in Materials and Methods, and is expressed as the mean value with the range in parentheses. Day-0 cells were obtained from ten subjects and day-3 macrophages were derived from five separate individuals Enzyme activity
Cells
1
2
3
L
I
I
I
I
5
6
I
I
kb
- 23
- 9.1 -6.5 - 1.L
pmol. min- ' mg protein-' Peripheral blood mononuclear cells (day 0) Monocyte-derived macrophages (day 3)
0.21 (0.07) 4.73 (2.2-8.8)
2.3
- 2.0 - 1.1 - 0.97 - 0.72
- 0.56
0
1
2 3 Days in culture
6
Fig. 4. Time course of induction of type 5 acid phosphatase in human monocyte-derived macrophages in culture. Peripheral blood mononuclear cells from a healthy individual were prepared as described in Materials and Methods and cultured with 10% autologous serum (reconstituted from citrated plasma by addition of 10mM calcium chloride) at 37°C. Adherent mononuclear cells were harvested at the indicated times and lysed in 50 mM Hepes/2% (massjvol.) Triton X-100 pH 7.5. Acid phosphatase activity in 32000xg supernatant fractions of the cell lysates was determined in duplicate by the immunoenzymatic procedure [24] and expressed as wmol 4-nitrophenol phosphate hydrolysed . rnin-' . mg protein-', measured by the dye-binding procedure [50].The data are representative of four similar experiments carried out with cells obtained from different individuals
spleen, bone marrow or reticulocytes. The greater expression of acid phosphatase RNA in macrophages, compared with monocytes from which these cells were derived, is consistent with the macrophage-specific nature of the enzyme. The increased expression also correlated closely with the data obtained by specific immunoenzymatic assay for type 5 acid phosphatase in these cells [24] (see Table 1). We observed very low activities of acid phosphatase in normal peripheral blood mononuclear cells and a greater than 20-fold increase after the cells had been cultured for three days in the presence of autologous serum. A time course (see Fig. 4) shows that expression of type 5 acid phosphatase activity reaches a peak at three days. It was found that the type 5 acid phosphatase constituted about one-half of the total acid phosphatase activity of the monocyte-derived macrophage. Acid phosphatase (type 5 isoenzyme) has been observed in the cells of hairy cell leukaemia, a B-cell disorder 1441which possesses, however, several other characteristics regarded as typical of macrophages. It is notable that a similar pattern of type 5 acid phosphatase expression can be induced when chronic lymphatic leukaemic cells, also of B-lineage (B-CLL),
Fig. 5. Genomic representation of type 5 acid phosphatase. Human genomic DNA was digested with XbaI (lanes 1 -4), EcoRI (lane 5), BamHI (lane 6 ) and probed with pPCR330. Lanes 1-4 depict two allelic restriction fragments: the DNA samples in lanes 3 and 4 were obtained from individuals that are homozygous and heterozygous respectively, for the least frequent allele that is associated with the 8-kb band. Size markers indicate fragments obtained from a mixture of A HindIII-digested DNA and pEMBL cut with TaqI
are exposed to phorbol ester [45]. This treatment induces transformation of B-CLL cells to hairy cells showing several phenotypic characteristics of macrophages [46]. Expression of certain other proteins, e.g. the mannose receptor, is tightly regulated in mononuclear cells and, like the type 5 acid phosphatase, is also stimulated by culture of peripheral blood monocytes in the presence of serum [47]. Although the mechanism by which regulation of macrophage-specific proteins occurs is unknown, the use of autologous serum culture of human monocytes should greatly facilitate study of the transcriptional control processes that are involved. Genomic representation and chromosomal localization of acid phosphatase
Human genomic DNA was digested with restriction endonucleases and probed with pPCR330. The patterns of hybridization seen in Southern blots of BamHI and EcoRI digests, consisting of single fragments of approximately 12 kb and 20 kb respectively, are consistent with the presence of a single copy gene. Digestion with XbaI revealed two allelic forms, the least frequent of which was detected in three out of eleven unrelated individuals (Fig. 5). The pHP3 probe far the human type 5 acid phosphatase gene hybridized intensely with a 6.7-kb BamHI fragment of mouse genomic DNA. Similar signals were detected in the rat (at 4.9 kb) and hamster (at 4.3 kb and 3.3 kb) digests, indicating that the acid phosphatase sequences are conserved between the human and rodent genomes.
292 Table 2. Concordance table f o r mappingpHP3 Human chromosome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16 ~~
Concordance -1Concordance+/+ Discordance-/+ Discordance+/-
=
28 6 7 1
31 3 4 4
22 5 13 2
31 3 4 4
28 4 7 3
27 3 8 4
30 3 5 4
27 3 8 4
31 1 4 6
24 4 I1 3
23 4 12 3
28 4 7 3
29 4
6 3
20 5 15 2
26 3 9 4
17
18
19
20
21
22
X
21 4 8 3
22 5 13 2
35 7 0 0
26 2 9 5
25 5 10 2
22 3 13 4
15 5 20 2
~
27 3 8 4
*10O I
13
14
15
16
17
18
19
20
21
22
X
Y
Fig. 6. Diagrammatic representation of frequency distribution of radioautographic grains over human chromosomes in 100 metuphase spread preparations. The peak frequency is at 19p 13.2 - 13.3
DNA prepared from a panel of 42 interspecific somatic cell hybrids was examined and the human 12-kb band was detected in seven (MOG34A4; LSR34+ S49; SIR74ii; SIR19A; POTB2/B2; HM76Dd; MOG2C2; data not shown). These positive hybrids have only human chromosome 19 in common. The segregation of pHP3 in the 42 interspecific somatic cell hybrids indicates that the gene may be assigned to chromosome 19 with zero discordance; there were at least eight examples of discordance with each of the other chromosomes, as summarized in Table 2. In situ hybridization was carried out using the probe pHP3 to confirm and refine the localization on human chromosome 19. In all, 199 silver grains were counted on chromosomes from 100 metaphase spreads and 24 grains were found on chromosome 19 (x’ = 25; P < 0.001). Of these 24 grains, 20 were present over chromosome bands 19p 13.2-13.3 with a peak at 19p 13.2 (Fig. 6). Recently human type 5 acid phosphatase has been reported to map to chromosome 15 by a newly described in situ hybridization method [48]. However, we have failed to confirm this localization either by investigation of an extensive panel of somatic cell hybrids (13 of which were discordant for chromosome 15) or by in situ hybridization (where no visible peak above background was observed for chromosome 1.5). The reason for this discrepancy is unclear, but our data using two different methods independently map the human acid phosphatase gene to human chromosome 19 and further indicate its sub-chromosomal locus at 19p 13.2-13.3. The identification of a frequent restriction fragment length polymorphism and this chromosomal localization in man confer on the type 5 acid phosphatase gene the potential for informative studies of human genetic linkage.
Two lysosomal enzymes (DNase and a-D-mannosidase B) as well as two important proteins that are expressed in monocyte-derived macrophages (the low-density lipoprotein receptor and third component of complement) also map to human chromosome 19p [49] (and references therein). In summary, we present the detailed characterization and mapping of the human type 5 acid phosphatase: a member of a unique family of metalloproteins. Purple acid phosphatases are conserved in mammals and possess multiple functions that include a role in porcine iron metabolism and a largely unknown role in relation to the differentiated cells of the mononuclear phagocytic system. Nonetheless, the specific expression of type 5 acid phosphatase that accompanies activation of human monocytes provides a unique focus for the study of macrophage transformation. We thank Dr Nigel Spurr at The Imperial Cancer Research Fund Laboratory, London and Dr S. Povey of The Galton Laboratory for kind provision of somatic cell hybrids. Mrs Joan Grantham kindly prepared the manuscript. Dr Janet Allen generously supplied materials and Dr John Savill helped to prepare human monocytes. This work was supported by The Cancer Research Campaign and, in part, by a grant from The Wellcome Trust.
REFERENCES 1. Roberts, R. M. & Bazer, F. W. (1980) in Steroid induced uterine proteins (Beato, M., ed.) pp. 133 - 149, Elsevier/North Holland, New York. 2. Antanaitis, B .C. & Aisen, P. (1983a) in Advunces in inorganic biochemistry (Theil, E. C., Eichhorn, G. L. & Marzilli, L., eds) vol.7, pp.111- 136, Elsevier, New York.
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29. Lin, C. C., Draqper, P. N., De Braekleer, M. (1985) Cytogenet. Cell Genet. 39, 269 - 274. 30. Harper, M. E. & Saunders, G. F. (1981) Chromosoma (Berl.) 83, 432 -439. 31. Wolff, S. & Perry, P. (1974) Chromosoma (Berl.) 48, 341 -353. 32. Lee, C. C., Wu, X., Gibbs, R. A., Cook, R. G., Muzny, D. M. & Caskey, C. T. (1988) Science239, 1288-1291. 33. Von Heijne, G. (1986) Nucleic Acids Res. 14, 4683 -4690. 34. Baumbach, G. A., Saunders, P. T. K., Bazer, F. W. & Roberts, R. M. (1984) Proc. Nut1 Acad. Sci. USA 81, 2985-2989. 35. Yeh, L-C., Lee, A. J., Lee, N. E., Lam, K-W. & Lee, J. C. (1987) Gene 60, 191- 196. 36. Pohlmann, R., Krentler, C., Schmidt, B., Schroder, W., Lorkowski, G., Culley, J., Mersmann, G., Geier, C., Waheed, A., Gottschalk, S., Grzeschik, K-H., Hasilik, A. & von Figura, K. (1988) EMBO J. 7,2343-2350. 37. Ketcham, C. M., Roberts, R. M., Simmen, R. C. M. & Nick, H. S. (1989) J . Biol. Chem. 264, 557-563. 38. Davis, J. C. & Averill, B. A. (1982) Proc. Natl Acad. Sci. U S A 79,4623 -4627. 39. Antanaitis, B. S. & Aisen, P. (1982) J. Biol. Chem. 257, 53305332. 40. Antanaitis, B. & Aisen, P. (1983b) in Structure and function of iron storage and transport proteins (Urushizaki, I., Listowsky, I. & Aisen P., eds) pp.503-511, Elsevier Science Publishers, North Holland, B.V. 41. Que, L. & Scarrow, R. C. (1988) Am. Chem. Soc. Symp. Ser. 372, 152-178. 42. Sjobert, B., Loehr, T. M. & Sanders-Loehr, J. (1982) Biochemistry 21,96-102. 43. Hayman, A. R., Warburton, M. J., Pringle, J. A. S., Coles, B. & Chambers, T. J. (1989) Biochem. J. 261,601 -609. 44. Korsmeyer, S. J., Greene, W. C., Cossman, J., Hsu, S. M., Jensen, J. P., Neckers, L. M., Marshall, S. L., Bakhshi, A,, Depper, J. M., Leonard, W. J., Jaffe, E. S. & Waldmann, T. A. (1983) Proc. Natl Acad. Sci. USA 80,4522-4525. 45. Totterman, T. H., Nilsson, K. & Sundstrom, C. (1980) Nature 288,176-178. 46. Caligaris-Cappio, F., Pizzolo, G., Chilosi, M., Bergui, L., Semenzato, G., Tesio, L., Morittu, L., Malavasi, F., Gobbi, M., Schwarting, R., Campana, D. & Janossy, G. (1985) BIood 66, 1035-1042. 47. Lennartz, M. R., Cole, F. S. & Stahl, P. D. (1989) J . Biol. Chem. 264,2385-2390. 48. Allen, B. S., Ketcham, C. M., Roberts, R. M., Nick, H. S. & Ostrer, H. (1989) Genomics 4, 597-600. 49. McKusick, V. A. (1988) Mendelian inheritance in man, 8th edn, John Hopkins University Press, Baltimore and London. 50. Bradford, M. M. (1976) Anal. Biochem. 72,248-254.
Type 5 acid phosphatase Sequence, expression and chromosomal localization of a differentiation-associated protein of the human macrophage Deirdre K. LORD', Nicholas C. P. CROSS', Maria A. BEVILACQUA', Susan H. RIDER3, Patricia A. GORMAN3, Ann V. GROVES3, Donald W. MOSS', Denise SHEER3, and Timothy M. COX' Department of Haemdtology, The Royal Postgraduate Medical School, London, England Department of Chemical Pathology, The Royal Postgraduate Medical School, London, England Cytogenics Laboratory, Imperial Cancer Research Fund Laboratories, London, England (Received October 4, 1989) - EJB 89 1203
The purple acid phosphatases and uteroferrin belong to a diverse multifunctional class of binuclear ironcontaining proteins that includes haemerythrin and ribonucleotide reductase. In the pig, uteroferrin has been implicated in the delivery of iron to the foetus, but the role of the related human type 5 acid phosphatase that is principally found in resident tissue macrophages is not yet clear. To define further the function of this metalloenzyme, we have isolated and sequenced a cDNA clone for type 5 acid phosphatase and investigated expression of its gene in human tissues. The phosphatase clone contains an open reading frame of 975 bp and encodes a protein of 325 amino acids, including a signal peptide of 19 residues and two potential sites for N-glycosylation. The type 5 acid phosphatase gene mapped to the short arm of human chromosome 19 and was found to have a restriction fragment length polymorphism on digestion with XbaI. Expression of phosphatase mRNA was restricted to mononuclear phagocytes and the enzyme was induced > 20-fold on transformation of normal human monocytes to macrophages by culture in serum-supplemented medium. Type 5 acid phosphatase thus represents a tightly regulated system for the study of molecular events in the differentiation programme of the normal macrophage. Type 5, tartrate-resistant, purple acid phosphatase is a basic, iron-binding protein with high activity towards phosphoproteins, ATP, and 4-nitrophenyl phosphate but with little action on aliphatic phosphates [I, 21. The isoenzyme is found in human alveolar macrophages [3] and osteoclasts [4] and is pathologically increased in Gaucher's cells [5] and in the hairy cells of leukaemic reticuloendotheliosis [6], now known as hairy cell leukaemia. It is thus expressed principally in cells of the human mononuclear macrophage system. Recently the protein has been purified from human and bovine spleen, where it is associated with a particulate fraction [7, 81. lmmunochemical and enzymatic studies [9, 101 as well as protein sequence analysis [I 11 have shown that the splenic enzyme is homologous to uteroferrin, a purple iron-binding protein abundant in the uterine secretions of pregnant sows. Physiological studies in vivo have implicated uteroferrin in the delivery of maternal iron to the developing piglet [12, 131 but the possibility of a similar function for the human tartrateresistant acid phosphatase has not been investigated. However, ultrastructural studies [14] indicate association of the bovine protein with ferritin and haemosiderin aggregates in splenic macrophages, suggesting that it may participate in the Correspondence to T. M . Cox, Dept. Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, England Note. The novel nucleotide sequence data published here has been deposited with EMBL sequence data bank and is available under accession number X14618. Enzymes. Type 5 acidphospkatase (EC 3.1.3.2); type I1 restriction endonucleases (EC 3.1.21.4).
retrieval of iron released by erythrophagocytosis. In addition, there is recent evidence that the enzyme is essential for bone resorption by human osteoclasts [15]. To investigate the function and expression of the type 5 acid phosphatase isoenzyme in human tissues, we have used a novel procedure to isolate a cDNA probe from human spleen and have utilized this to characterize full-length clones from a human cDNA library. We have further utilized the full-length cDNA to monitor expression of type 5 acid phosphatase in human tissues and to determine the chromosomal localization of its gene. MATERIALS AND METHODS cDNA amplification
Poly-(A)-rich RNA, purified from Gaucher's spleen, was reverse-transcribed using an oligo(dT) primer [16]. The RNA was hydrolysed by addition of NaOH to 66mM and incubating at 65 "C for 1 h. After neutralization with HCI in the presence of 0.2M Tris, the single-stranded cDNA was ethanolprecipitated and resuspended in 10 mM Tris/HCl, 0.1 mM EDTA pH 8.0. Published amino acid sequences for porcine uteroferrin and acid phosphatase from bovine spleen [ l l ] were used to derive oligonucleotide primers for use in the polymerase chain reaction to amplify human phosphatase sequences from the single-stranded cDNA. Oligonucleotides were synthesized to provide all possible sequences coding for two regions conserved between porcine uteroferrin and bovine spleen phos-
288 phatase. Sense oligonucleotides were made to FQETFED and antisense to GHYPVW. Each pool of oligonucleotide primers was highly degenerate and contained 256 individual sequences. The polymerase chain reaction was carried out using 100 ng cDNA and 2.5 U Taq polymerase (Perkin Elmer Cetus), as recommended by the manufacturer, except that the primer concentration was increased to 10 pM. The reaction was carried out in a home-made thermocycling apparatus 1171 under the following conditions: 92"C, 2 min; 45"C, 2 min; 72"C, 2 min. After 30 cycles, a final extension of 10 min at 72°C was made and an aliquot of the reaction mixture was analysed on a 2% agarose minigel. Amplified DNA was gelpurified by the freeze-squeeze technique [18], treated with polynucleotide kinase [16]and ligated into pEMBL19 cut with Smal and dephosphorylated.
trifugation through discontinuous plasma/Percoll gradients, as described by Savill et al. [23]. The upper layer of mononuclear cells was removed from the underlying polymorphonuclear cells, washed and resuspended at 4 x lo6 cells/ml in Iscove's modified Dulbecco's medium. 1ml was added to each well of 24-well tissue culture plates. After a 1 h incubation at 37°C and 6% C 0 2 , the wells were washed and adherent monocytes cultured for three days in Iscove's medium with 10% autologous serum. Assay of type 5 human acidphosphatase Type 5 tartrate-resistant acid phosphatase activity was assayed using the immunoenzymatic method described by Whitaker et a1 1241with 4-nitrophenyl phosphate as substrate. Chromosomal mapping
cDNA library screening A 1" g t l l cDNA library (Clontech) made from human placenta was used for screening. Plaque and colony hybridization was carried out according to Maniatis et al. [16]. CDN A probes cDNA probes were labelled with [ W ~ ~ P I ~ C using T P the multiprime DNA-labelling system (Amersham). D N A sequencing Restriction fragments were subcloned into pEMBL 18 or 19 and both strands were sequenced by the chain-terminator method [I91 using the M13 universal primer (New England Biolabs). The cloning and sequencing strategy is shown in Fig. 1. Southern blot analysis DNA was extracted from peripheral blood samples by urea/SDS lysis [20], digested, electrophoresed in an 0.8% agarose gel and then transferred to Hybond N membrane (Amersham) as described by Southern [21]. Hybridization with the phosphatase probe was carried out at 65°C in 6 x NaCl/Cit (NaCl/Cit: 0.15 M NaC1/15 mM trisodium citrate, pH 7.5) 0.1% SDS, 5 x Denhardt's, 100 pg/ml salmon sperm DNA for 16 h. Filters were washed in 1 x NaCI/Cit 0.1 % SDS and 0.2 x NaCl/Cit 0.1 % SDS at 65 "C for 1 h. Northern blot analysis Total RNA was prepared from human tissues by the guanidinium isothiocyanate method [22] and quantified by ultraviolet spectroscopy [16]. 10-pg samples were electrophoresed on 1.5% formaldehyde/agarose gels, transferred to Hybond N membranes (Amersham) and hybridized at 42 "C in 5 x Denhardt's solution, 6 x NaCl/Cit, 0.5% SDS, 50% formamide, 100 pg/ml salmon sperm DNA for 16 h. The filters were washed once in 10 x Denhardt's, 6 x NaCl/Cit, 0.1 % SDS and then twice in 3 x NaCl/Cit, 0.1% SDS at 65°C for 30 min.
Cells and D N A preparation. Cell lines were maintained in RPMI 1640 medium with 10% foetal calf serum at 37°C under an atmosphere of 95% 02/5% C 0 2 . To retain human chromosome 17 or X, where appropriate, media were supplemented with 100 pM hypoxanthine, 10 pM methotrexate and 10 pM thymidine. All 42 hybrid cell lines have been previously described (references available on request from DKL) and their chromosome content was defined by enzymatic and karyotypic analysis. Assays for human enzymes were carried out using standard methods [25] and the karyotypes were determined by a combination of cell staining and quinacrine banding [26, 271. Approximately 5 x lo7 fresh cells were used for extraction of high-molecular-mass genomic DNA [28]. 30 pg hybrid DNA, parental rodent DNA and 15 pg human DNA (obtained from fresh lymphocytes) were digested with BamHI (BRL) and electrophoresed for Southern blotting as described above. A 1.4-kb EcoRI fragment, representing a full-length cDNA was used to probe the phosphatase gene after hybridization to high stringency with 0.1 x NaCl/Cit, 0.1% SDS at 65 "C. The washed filters were exposed to XAR5 film (Kodak) with intensifying screens (Dupont) at - 70°C for up to seven days. In situ hybridization Human lymphocytes were cultured with phytohaemagglutinin for 72 h at 37°C and bromodeoxyuridine was added to give a final concentration of 200 pg/ml [29]. After 16 h, the cells were washed and further incubated in thymidinesupplemented medium for 6 h. Cell harvesting and all further procedures were carried out in subdued lighting. In situ hybridization was based on the method of Harper and Saunders [30] using acid phosphatase cDNA labelled with [3H]deoxyribonucleotidesto a specific activity of 3 x 10' cpm/ pg. The hybridization was carried out at 37°C with probe concentrations of 0.02 pg/ml and 0.1 pg/ml. Slides were washed in 2 x NaCl/Cit at 39"C, dehydrated and coated with Ilford K5 emulsion. Slides were developed after 6 - 10 days and G-banded by a procedure based on the method of Wolff and Perry [31] RESULTS AND DISCUSSION
Preparation of monocyte-derived macrophages
Cloning of tartrate-resistant phosphatase
Mononuclear cells were isolated from citrated peripheral blood by a combination of dextran sedimentation and cen-
Human acid phosphatase was cloned by a novel procedure that utilizes the polymerase chain reaction to amplify cDNA
289
c
2
wl
a W
4
h
-
-+--c---
Fig 1 . Restriction map and sequencing strategy for the tartrate-resistant acidphosphatase cDNA clone HP3. The position of restriction sites used in sequencing are indicated. The solid box represents the coding region, the lines designate the 5’- and 3’-untranslated regions. Arrows show the direction and the extent of each sequence determination
using pools of oligonucleotide primers designed on the basis of the protein sequence [32]. Specifically, we selected regions shared between bovine acid phosphatase and porcine uteroferrin and we synthesized mixed oligonucleotides with a high degree of degeneracy. In uteroferrin the distance between these sequences is 115 amino acid residues, but attempts to amplify the human gene from genomic DNA were unsuccessful, presumably because of the presence of one or more introns in this region. Thus single-stranded cDNA was used as the template for the reaction. The RNA used for reverse transcription was obtained from the spleen of a patient with Gaucher’s disease, a disorder in which expression of type 5 acid phosphatase is enhanced. Analysis of the reaction products after amplification showed a major fragment of 330 bp, i.e. approximately the predicted size. The 330-bp DNA was cloned to yield the plasmid pPCR330, the insert of which was sequenced. The derived amino acid sequence of this clone was found to be highly homologous to the corresponding sequence of porcine uteroferrin and bovine spleen acid phosphatase. pPCR330 was used to screen 1.25 x l o 5 plaques of a human placental cDNA library in A g t l l . Three putative clones were plaque-purified, digested with EcoRI and found to contain 1.3- 1.4-kb inserts. The insert from the largest of these (A HP3) was subcloned and sequenced following the strategy shown in Fig. 1. Fig. 2 shows the 1359-nucleotide sequence and deduced amino acid sequence of the human type 5 acid phosphatase. Acid phosphatase cDNA comprises a 35-bp 5’-untranslated region followed by one of two possible in-frame initiator methionines both of which lie downstream of an in-frame stop codon at nucleotide 12-14. The open reading frame is 975 bp and encodes 325 amino acids that correspond to a polypeptide of molecular mass 36571 Da. The 3’-non-coding region is 346-bp long with a potential polyadenylation signal 19 bp upstream of the poly(A) tract. The 5‘ end of one of the smaller cDNA insert HP1 was sequenced and found to be identical to HP3 but started slightly downstream at nucleotide 186. The nucleotide sequence of full-length acid phosphatase is identical to that of pPCR330, except for the replacement of C at positions 424 and 519 by G. This discrepancy between the spleen and placental sequences may represent allelic variations or reflect replication errors by Tuq polymerase during the polymerase chain reaction. The NH,-terminal region of acid phosphatase contains many hydrophobic residues that are characteristic of a signal peptide. The assignment of the cleavage site between resi-
dues -1 and + l is based on Von Heijne’s (-3,-I) rule [33]. However, this remains tentative until the NH,-terminal residue of the mature protein is known. The sequence predicts two possible glycosylation sites (Asn-Val-Ser) for asparaginelinked oligosaccharides at residues 97 and 128. Type 5 human acid phosphatase, bovine spleen phosphatase and uteroferrin are homologous proteins with similar physical and enzymatic properties. Moreover, comparative partial sequence studies have confirmed that there is greater than 75% conservation at the amino acid level between bovine spleen phosphatase and porcine uteroferrin [l 11. In common with the type 5 phosphatases, uteroferrin possesses acid phosphatase activity but it appears to be a predominantly secreted protein, whereas the type 5 acid phosphatase is found within cells and has been localized to membrane-bound organelles. The mannose 6-phosphate residue of uteroferrin has been shown in biosynthetic labelling experiments to be masked, and this may explain its extrusion by endometrial cells in culture [34]. Comparison of the predicted amino acid sequence of the human type 5 phosphatase with the human prostatic [35] and lysosomal [36] acid phosphatases revealed no sequence similarity and additional searches have failed to show any significant resemblance to other known lysosomal enzymes. Computer-assisted analysis did not reveal an obvious hydrophobic membrane-spanning domain. Since completing the sequence of our cDNA clones, Ketcham et al. have published a sequence of type 5 acid phosphatase from human placenta [37]. There are four discrepancies between the sequence reported here and that published by Ketcham et al. Two residues (Lysl60 and Leul61) were found in this study, in the phosphatase clone from human spleen and in the human placental sequence, that are not present in Ketcham’s sequence, which predicts a protein that is two amino acids shorter. However, because our sequence was found in cDNA from two independent sources, we believe it to be correct. We also found Ala and Arg at positions 26 and 27 of the protein where Gly and Pro were reported by Ketcham et al. The explanation for this is unclear but these discrepancies arise in a region found to be subject to sequence compression that we resolved by the use of ITP in the sequencing reactions. Moreover, the sequence contains a SmaI site that was used in cloning (see Fig. 1). Previous studies have indicated that iron, which is essential for the enzymatic activity of uteroferrin and spleen acid phosphatase, is present in these proteins as an anti-ferromagnetically coupled binuclear p o x 0 core [38]. The mixed valence ferric-ferrous couple of the pink, reduced form of these pro-
290 AGAGCCTCCGGTGACTGGCCTGTGTCTCCCCCTGG --19 -10 Met Asp Met Trp Thr Ala Leu Leu Ile Leu Gln Ala Leu Leu Leu ATG GAC ATG TGG ACG GCG CTG CTC ATC CTG CAA GCC TTG TTG CTA
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Ile Ala Tyr Ser Lys Ile Ser Lys Arg Trp Asn Phe Pro Ser Pro ATT GCA TAC TCT AAG ATC TCC AAG CGC TGG AAC TTC CCC AGC CCT 120 130 Phe Tyr Arg Leu H i s Phe Lys Ile Pro Gln Thr Asn Val Ser Val TTC TAC CGC CTG CAC TTC AAG ATC CCA CAG ACC AAT GTG TCT GTG
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Glu Met Thr Val Thr Tyr Ile Glu Ala Ser Gly Lys Ser Leu Phe GAG ATG ACT GTC ACT TAC ATC GAG GCC TCG GGC AAG TCC CTC TTT 980 300 Lys Thr Arg Leu Pro Arg Arg Ala Arg Pro * AAG ACC AGG CTG CCG AGG CGA GCC AGG CCC TGA ACTCCCATGACTGCC 1028 CAGCTCTGAGGCCCGATCTCCACTGTTGGGTGGGTGGCCTGCCGGGACCCTGCTCACAGG 1088 CAGGCTTTTCCTCCAACCTGTGGCGCTGCAGCAGGGCAGGAAGGGGAAACACAGCTGATG 1148 RACTGTGGTGCCACATGACCCTTGTGGCACAGATGCCCACGTATGTGAAACACACATGGA 1208 CATGTGTCCCAGCCACAGTGTTATGCTCTGTCGCTGGCTGGCTCACCTTTGCTGAGTTCCGGGG 1268 TGCAATGGGGGAGGGAGGGAGGGAAAGCTTCCTTCCTCCT~TCAAGCATCTTTCTGTTACTG 1328 ATGTTC-GAATAGTTGCCAAGGCTG
1359
Fig 2. Nucleotide and deduced amino acid sequence of human tartrateresistant acid phosphatase cDNA. The signal peptidase cleavage site follows amino acid - 1, and the + 1 amino acid is assigned to the first residue of the mature protein. The two possible N-linked glycosylation sites (Asn-Val-Ser) are at residues 97-99 and 128- 130. The termination codon is indicated by an asterisk and the putative polyadenylation signal in the 3’-untranslated region is underlined
teins generates an intense EPR signal at g’ = 1.74 [39, 401. An almost identical signal has been detected in other ironcontaining proteins, e.g. ribonucleotide reductase [41], and this feature appears to be diagnostic of a unique class of metalloproteins. Although the binuclear iron-containing proteins are characterized by specific EPR signals, with the exception of their unique iron centre, they show marked divergence in their amino acid sequences, which probably reflects their different functions. On the other hand, the purple acid phosphatases and uteroferrin show remarkable sequence conservation, which suggests a common functional role. There is evidence that histidine and tyrosine residues are involved in the binding of the two iron atoms [2, 401 (and
Fig. 3. Northern blot analysis of 10-pg aliquots of RNA from human tissues and cells using full-length acid phosphatase cDNA as a probe. Total RNA was isolated from Gaucher’s spleen (lane l), normal spleen (lane 2), bone marrow (lane 3), reticulocytes (lane 4), lymphocytes (lane 5), liver (lane 6), monocytes (lane 7) and monocyte-derived macrophages (lane 8). A 1.5-kb mRNA for acid phosphatase is seen in samples from Gaucher’s disease spleen and macrophages. Markers at the right of the figure show the position of 28s and 18s rRNA. The exact size of the acid phosphatase transcript was determined using RNA calibration standards (BRL)
references therein), and a search for amino acid repeats in human acid phosphatase revealed a His/Tyr cluster at positions 86-94 and an almost exact mirror-image sequence at positions 221 - 227, which may thus constitute iron-binding domains. In ribonucleotide reductase, the active centre tyrosine at position 122 is found in conjunction with a highlyconserved histidine residue at position 118 [42]; a configuration that is conserved between uteroferrin, bovine spleen phosphatase and human type 5 acid phosphatase at positions 223 (His) and 227 (Tyr). This region appears to be the most plausible candidate for the iron-binding site, but the occurrence of two similar domains in human acid phosphatase raises the possibility that four rather than two iron atoms may be bound for each polypeptide, as suggested by analysis of the enzyme isolated from human osteoclasts [43]. Expression of type 5 acidphosphatase in human tissues RNA from a range of human tissues and cells was probed with the labelled cDNA insert from pHP3. RNA with a length of 1.5 kb was found to be expressed in large amounts in Gaucher’s spleen and macrophages (see Fig. 3). A barely detectable signal was seen in resting monocytes, liver and lymphocytes and none was detected in total RNA from control
29 1 Table 1. Type 5 acidphosphatase in human monocytes andmacrophages Type 5 acid phosphatase activity was determined immunoenzymatically as described in Materials and Methods, and is expressed as the mean value with the range in parentheses. Day-0 cells were obtained from ten subjects and day-3 macrophages were derived from five separate individuals Enzyme activity
Cells
1
2
3
L
I
I
I
I
5
6
I
I
kb
- 23
- 9.1 -6.5 - 1.L
pmol. min- ' mg protein-' Peripheral blood mononuclear cells (day 0) Monocyte-derived macrophages (day 3)
0.21 (0.07) 4.73 (2.2-8.8)
2.3
- 2.0 - 1.1 - 0.97 - 0.72
- 0.56
0
1
2 3 Days in culture
6
Fig. 4. Time course of induction of type 5 acid phosphatase in human monocyte-derived macrophages in culture. Peripheral blood mononuclear cells from a healthy individual were prepared as described in Materials and Methods and cultured with 10% autologous serum (reconstituted from citrated plasma by addition of 10mM calcium chloride) at 37°C. Adherent mononuclear cells were harvested at the indicated times and lysed in 50 mM Hepes/2% (massjvol.) Triton X-100 pH 7.5. Acid phosphatase activity in 32000xg supernatant fractions of the cell lysates was determined in duplicate by the immunoenzymatic procedure [24] and expressed as wmol 4-nitrophenol phosphate hydrolysed . rnin-' . mg protein-', measured by the dye-binding procedure [50].The data are representative of four similar experiments carried out with cells obtained from different individuals
spleen, bone marrow or reticulocytes. The greater expression of acid phosphatase RNA in macrophages, compared with monocytes from which these cells were derived, is consistent with the macrophage-specific nature of the enzyme. The increased expression also correlated closely with the data obtained by specific immunoenzymatic assay for type 5 acid phosphatase in these cells [24] (see Table 1). We observed very low activities of acid phosphatase in normal peripheral blood mononuclear cells and a greater than 20-fold increase after the cells had been cultured for three days in the presence of autologous serum. A time course (see Fig. 4) shows that expression of type 5 acid phosphatase activity reaches a peak at three days. It was found that the type 5 acid phosphatase constituted about one-half of the total acid phosphatase activity of the monocyte-derived macrophage. Acid phosphatase (type 5 isoenzyme) has been observed in the cells of hairy cell leukaemia, a B-cell disorder 1441which possesses, however, several other characteristics regarded as typical of macrophages. It is notable that a similar pattern of type 5 acid phosphatase expression can be induced when chronic lymphatic leukaemic cells, also of B-lineage (B-CLL),
Fig. 5. Genomic representation of type 5 acid phosphatase. Human genomic DNA was digested with XbaI (lanes 1 -4), EcoRI (lane 5), BamHI (lane 6 ) and probed with pPCR330. Lanes 1-4 depict two allelic restriction fragments: the DNA samples in lanes 3 and 4 were obtained from individuals that are homozygous and heterozygous respectively, for the least frequent allele that is associated with the 8-kb band. Size markers indicate fragments obtained from a mixture of A HindIII-digested DNA and pEMBL cut with TaqI
are exposed to phorbol ester [45]. This treatment induces transformation of B-CLL cells to hairy cells showing several phenotypic characteristics of macrophages [46]. Expression of certain other proteins, e.g. the mannose receptor, is tightly regulated in mononuclear cells and, like the type 5 acid phosphatase, is also stimulated by culture of peripheral blood monocytes in the presence of serum [47]. Although the mechanism by which regulation of macrophage-specific proteins occurs is unknown, the use of autologous serum culture of human monocytes should greatly facilitate study of the transcriptional control processes that are involved. Genomic representation and chromosomal localization of acid phosphatase
Human genomic DNA was digested with restriction endonucleases and probed with pPCR330. The patterns of hybridization seen in Southern blots of BamHI and EcoRI digests, consisting of single fragments of approximately 12 kb and 20 kb respectively, are consistent with the presence of a single copy gene. Digestion with XbaI revealed two allelic forms, the least frequent of which was detected in three out of eleven unrelated individuals (Fig. 5). The pHP3 probe far the human type 5 acid phosphatase gene hybridized intensely with a 6.7-kb BamHI fragment of mouse genomic DNA. Similar signals were detected in the rat (at 4.9 kb) and hamster (at 4.3 kb and 3.3 kb) digests, indicating that the acid phosphatase sequences are conserved between the human and rodent genomes.
292 Table 2. Concordance table f o r mappingpHP3 Human chromosome
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16 ~~
Concordance -1Concordance+/+ Discordance-/+ Discordance+/-
=
28 6 7 1
31 3 4 4
22 5 13 2
31 3 4 4
28 4 7 3
27 3 8 4
30 3 5 4
27 3 8 4
31 1 4 6
24 4 I1 3
23 4 12 3
28 4 7 3
29 4
6 3
20 5 15 2
26 3 9 4
17
18
19
20
21
22
X
21 4 8 3
22 5 13 2
35 7 0 0
26 2 9 5
25 5 10 2
22 3 13 4
15 5 20 2
~
27 3 8 4
*10O I
13
14
15
16
17
18
19
20
21
22
X
Y
Fig. 6. Diagrammatic representation of frequency distribution of radioautographic grains over human chromosomes in 100 metuphase spread preparations. The peak frequency is at 19p 13.2 - 13.3
DNA prepared from a panel of 42 interspecific somatic cell hybrids was examined and the human 12-kb band was detected in seven (MOG34A4; LSR34+ S49; SIR74ii; SIR19A; POTB2/B2; HM76Dd; MOG2C2; data not shown). These positive hybrids have only human chromosome 19 in common. The segregation of pHP3 in the 42 interspecific somatic cell hybrids indicates that the gene may be assigned to chromosome 19 with zero discordance; there were at least eight examples of discordance with each of the other chromosomes, as summarized in Table 2. In situ hybridization was carried out using the probe pHP3 to confirm and refine the localization on human chromosome 19. In all, 199 silver grains were counted on chromosomes from 100 metaphase spreads and 24 grains were found on chromosome 19 (x’ = 25; P < 0.001). Of these 24 grains, 20 were present over chromosome bands 19p 13.2-13.3 with a peak at 19p 13.2 (Fig. 6). Recently human type 5 acid phosphatase has been reported to map to chromosome 15 by a newly described in situ hybridization method [48]. However, we have failed to confirm this localization either by investigation of an extensive panel of somatic cell hybrids (13 of which were discordant for chromosome 15) or by in situ hybridization (where no visible peak above background was observed for chromosome 1.5). The reason for this discrepancy is unclear, but our data using two different methods independently map the human acid phosphatase gene to human chromosome 19 and further indicate its sub-chromosomal locus at 19p 13.2-13.3. The identification of a frequent restriction fragment length polymorphism and this chromosomal localization in man confer on the type 5 acid phosphatase gene the potential for informative studies of human genetic linkage.
Two lysosomal enzymes (DNase and a-D-mannosidase B) as well as two important proteins that are expressed in monocyte-derived macrophages (the low-density lipoprotein receptor and third component of complement) also map to human chromosome 19p [49] (and references therein). In summary, we present the detailed characterization and mapping of the human type 5 acid phosphatase: a member of a unique family of metalloproteins. Purple acid phosphatases are conserved in mammals and possess multiple functions that include a role in porcine iron metabolism and a largely unknown role in relation to the differentiated cells of the mononuclear phagocytic system. Nonetheless, the specific expression of type 5 acid phosphatase that accompanies activation of human monocytes provides a unique focus for the study of macrophage transformation. We thank Dr Nigel Spurr at The Imperial Cancer Research Fund Laboratory, London and Dr S. Povey of The Galton Laboratory for kind provision of somatic cell hybrids. Mrs Joan Grantham kindly prepared the manuscript. Dr Janet Allen generously supplied materials and Dr John Savill helped to prepare human monocytes. This work was supported by The Cancer Research Campaign and, in part, by a grant from The Wellcome Trust.
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