Proc. Nati. Acad. Sci. USAVol. 83, pp.' 7182-7186, October 1986Biochemistry

Isolation and characterization of a cDNA encoding a humanliver/bone/kidney-type alkaline phosphatase

(cDNA expression library/DNA sequence analysis/isoenzymes/osteosarcoma cells)MITCHELL J. WEISS*, PAULA S. HENTHORN*t, MARY ANN LAFFERTY*, CLIVE SLAUGHTERS,MICHAEL RADUCHA*, AND HARRY HARRIS**Department of Human Genetics, University of Pennsylvania School of Medicine, and tDivision of Biochemical Development and Molecular Diseases,Children's Hospital of Philadelphia, Philadelphia, PA 19104; and *Department of Pathology and Laboratory Medicine,University of Texas Medical School, Houston, TX 77225Contributed by Harry Harris, June 10, 1986

ABSTRACT Alkaline phosphatases (ALPs) [orthophos-phoric-monoester phosphohydrolase (alkaline optimum), EC3.1.3.1] isolated from human liver, bone, and kidney (L/B/K)exhibit very similar biochemical and immunologic propertiesthat differentiate them from other human ALPs, such as thosecharacteristically found in placenta and intestine. Despite theirsimilarities, the L/B/K ALPs produced in different tissuesshow slight physical differences. To examine structural andevolutionary relationships between the various ALPs, a cDNAcorresponding to L/B/K ALP mRNA has been isolated. AAgtlI cDNA expression library was constructed using poly(A)RNA from the osteosarcoma cell line Saos-2 and screened withanti-liver ALP antiserum. The 2553-base-pair cDNA containsan open reading frame that encodes a 524 amino acid poly-peptide with a predicted molecular mass of 57.2 kDa. This ALPprecursor protein contains a presumed signal peptide of 17amino acids followed by 37 amino acids that are identical to theamino-terminal sequence determined from purified liver ALP.In addition, amino acid sequences of several CNBr peptidesobtained from liver ALP are found within the cDNA-encodedprotein. The deduced L/B/K ALP precursor polypeptideshows 52% homology to human placental ALP and 25%homology to Escherichia coli ALP precursor polypeptides.Sixty percent nucleotide homology exists between the humanL/B/K and placental cDNAs over the protein coding regions.The 5' and 3' untranslated regions of the L/B/K ALP cDNA,176 and 805 base pairs, respectively, show no homology to thecorresponding regions of placental ALP cDNA.

Alkaline phosphatases (ALPs) [orthophosphoric-monoesterphosphohydrolase (alkaline optimum), EC 3.1.3.1] hydrolyzevarious monophosphate esters at a high pH optimum. Theseenzymes, which are widely distributed in nature, exist asmembrane-bound glycoproteins in higher organisms (1). Theenzyme products of at least three ALP gene loci [placental,intestinal, and liver/bone/kidney (L/B/K)] are distinguish-able in man by a variety of structural, biochemical, andimmunologic methods (2-5). The placental and intestinalforms of human ALP are expressed in a relatively tissue-specific manner in normal individuals. In contrast, L/B/KALP is expressed at relatively high levels in osteoblasts andto a lesser extent in fibroblasts, leukocytes, and cells fromnumerous other tissues, including liver, kidney, breast, andbrain (1, 2). Slight differences in electrophoretic mobility andthermostability between the L/B/K ALPs from varioustissues are attributed to differences in posttranslationalmodification, although it is possible that their protein moi-eties are encoded by separate but related genes (2). Little isknown regarding the physiological function ofALPs in most

tissues except that the bone isoenzyme has long been thoughtto have a role in normal skeletal mineralization (6).

In this report, we describe the isolation and nucleotidesequence analysis of a cDNA corresponding to a humanL/B/K ALP. This cDNA probably represents ALP of boneorigin since it was derived from Saos-2 osteosarcoma cellsthat have osteoblastic properties (7). Partial amino acidsequences obtained from purified liver ALP are identical toportions of the polypeptide encoded by the L/B/K cDNA.This is consistent with the hypothesis that the ALPs found inliver and bone are identical at the amino acid level. Compar-ison of the protein sequences of human L/B/K, humanplacental, and Escherichia coli ALPs shows conservation ofprimary structure in catalytically important regions.

MATERIALS AND METHODS

Purification of Liver ALP. Human liver ALP was purifiedto homogeneity using an immunoaffinity column made withmonoclonal antibody ALPp/Sp2/19 as described (8).

Antiserum. Anti-human liver ALP antiserum was raised ina rabbit using purified liver ALP as antigen.

Protein Sequence Determination. Amino acid sequenceanalysis of purified liver ALP and its CNBr peptides wasperformed as described (9).

Cell Lines. The Saos-2 cell line, originally obtained fromthe laboratory of Jorgen Fogh (10), is derived from a humanosteogenic sarcoma. These cells have been shown to producerelatively large amounts of ALP of the L/B/K type (11).

Isolation of Poly(A) RNA from Saos-2 Cells. Total cellularRNA was isolated from Saos-2 cells using the guanidin-ium/cesium chloride method (12). Poly(A) RNA was isolatedby two rounds of oligo(dT)-cellulose chromatography (12).cDNA Library Construction. Construction of a cDNA

library using the bacteriophage expression vector Xgtll wascarried out as described (9). Double-stranded cDNA (0.02 ,ug)was ligated to 0.5 ug of Xgtll arms (Vector Cloning Systems,San Diego, CA) and packaged into phage heads. The result-ant library of 3 x 10' recombinant phage was amplified on E.coli strain Y1088.

Screening cDNA Libraries with Antibody. The Saos-2cDNA library was screened with rabbit anti-human liverALPantiserum according to Young and Davis (13). Bound anti-body was detected using an avidin-biotin system (VectastainABC Kit, Vector Laboratories, Burlingame, CA).

Nucleic Acid Hybridization Analysis. Radiolabeled DNAprobes were prepared either by nick-translation (Nick-Trans-lation Kit, Bethesda Research Laboratories) or calf thymus

Abbreviations: ALP, alkaline phosphatase; kb, kilobase pair(s); bp,base pair(s); L/B/K, liver/bone/kidney.To whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 83 (1986) 7183

DNA primer labeling (14). RNA was separated by electro-phoresis in 1% agarose gels containing 2.2 M formaldehyde(12). DNA was fractionated in 0.8% agarose/Tris borate gels(12). Nucleic acids were transferred to Zetabind membrane(AMF Specialty Materials Group, Meriden, CT) and hy-bridized to 32P-radiolabeled probes according to the manu-facturer's instructions. The membranes were washed understringent conditions (30 mM NaCl/3 mM sodium citrate for1-2 hr at 680C) and autoradiographed.

Hybrid-Selected Translation. Twenty micrograms ofcDNAin plasmid vector pAT153 was linearized with EcoRI, dena-tured, and immobilized on a 0.6-cm square of nitrocellulosepaper (15). Hybridization to 15 ,ug of Saos-2 poly(A) RNA,washing of the filter, and subsequent elution ofRNA were asdescribed (12), except that RNA was eluted in a step-wisefashion at 70, 80, and 1000C. The RNA was isolated byethanol precipitation and in vitro translation was performedusing a rabbit reticulocyte lysate system (Bethesda ResearchLaboratories) with [35S]methionine according to the manu-facturer's instructions. Immunoprecipitation ofin vitro trans-lated protein was performed according to published proce-dures (16, 17). Polypeptides were fractionated on 10% poly-acrylamide gels under denaturing conditions (18).DNA Sequence Analysis. The 2553-base-pair (bp) L/B/K

ALP cDNA was sequenced by the Sanger dideoxy chain-termination method (19). Overlapping M13 subclones weregenerated by using the T4 polymerase deletion method ofDale et al. (20) and by subcloning various restriction frag-ments into M13. Both strands of the cDNA were sequencedin full. Computer analyses of DNA and protein sequenceswere performed using the IBI/Pustell DNA and ProteinSequence Analysis System (International Biotechnologies,New Haven, CT), FASTP (21), and NUCALN (22).

RESULTS

Immunoprecipitation ofSaos-2 in Vitro Translated Products.Saos-2 poly(A) RNA was translated in vitro using a rabbitreticulocyte lysate system. The resultant polypeptides wereimmunoprecipitated with rabbit anti-liver ALP antiserum.NaDodSO4/polyacrylamide gel analysis of the immunopre-

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cipitates is shown in Fig. 1A. A single 58-kDa band isprecipitated by anti-liver ALP and not by preimmune serum.The size of this polypeptide is consistent with the expectedsize of unglycosylated L/B/K ALP precursor protein (23).

Isolation of a L/B/K ALP cDNA from Saos-2 cDNA. TheSaos-2 cDNA library was screened with anti-liver ALPantiserum. Seven immunoreactive clones were isolated fromscreening 5 x 105 recombinant phage. The cDNA insertswere subcloned into pAT153 and examined by DNA blothybridization analysis using the various candidate cDNAinserts as hybridization probes. Four of the clones were ofidentical size [2.5 kilobase pairs (kb)], hybridized to eachother, and reacted most intensely with the antiserum. Ahybrid-selection translation assay was performed to verify thatthese 2.5-kb cDNAs encode ALP. A plasmid subclone of oneof the 2.5-kb cDNA inserts, pS3-1, was used to select cross-hybridizing nucleic acid from Saos-2 poly(A) RNA. Fig. 1B andC show that the RNA selected by pS3-1 programed thesynthesis of a 58-kDa protein that was immunoprecipitable byliver ALP antiserum. RNA blot analysis reveals that pS3-1hybridizes to a single 2.6-kb band in Saos-2 RNA (data notshown), indicating that the cDNA is nearly full length.DNA Sequence Analysis. The nucleotide sequence of the

cDNA insert of pS3-1 was determined (Fig. 2). The sequencecontains an open reading frame, beginning at nucleotide 177,that encodes a 524 amino acid polypeptide. Nucleotidessurrounding the AUG initiation codon agree at all but the lastposition with a consensus sequence, ACCAUGG, that hasbeen determined by examination of translation initiation sites(24, 25). The predicted molecular mass of this polypeptide,57.2 kDa, agrees with the 58-kDa polypeptide obtained byimmunoprecipitation of in vitro translation products directedby Saos-2 RNA.We and others (23, 26) have determined the sequence of 37

amino acid residues from the amino terminus of purified liverALP. An additional 50 residues of liver ALP were determinedfrom four CNBr peptides. These sequences of liver ALP (37amino-terminal residues and 50 internal CNBr residues) areidentical to segments of the polypeptide sequence encoded bythe pS3-1 cDNA insert and are underlined in Fig. 2.The polypeptide encoded by the L/B/K ALP cDNA

C

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FIG. 1. (A) Immunoprecipitation of Saos-2 RNA in vitro translation products. Saos-2 poly(A) RNA was translated in a rabbit reticulocytelysate in vitro translation system, immunoprecipitated with either rabbit anti-liver ALP antiserum (aLALP) or preimmune serum, andfractionated by NaDodSO4/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). (B) Hybrid-selected translation of Saos-2 poly(A) RNA.Saos-2 RNA was hybrid-selected with pS3-1 and the plasmid vector pAT153. RNA that hybridized to the above plasmids was eluted at 70, 80,and 100C prior to in vitro translation and NaDodSO4/PAGE. (C) Immunoprecipitation of hybrid-selected translation products from B. The invitro translation products of Saos-2 RNA that had been hybrid-selected by pS3-1 or pAT153 were immunoprecipitated by anti-liver ALPantiserum and fractionated by NaDodSO4/PAGE. Molecular masses are shown in kDa.

ae) pS3-1 pAT 153

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Biochemistry: Weiss et al.

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7184 Biochemistry: Weiss et al. Proc. Natl. Acad. Sci. USA 83 (1986)

1 CGCGCCCGCTATCCTGGCTCCGTGCTCCCACGCGCTTGTGCCTGGACGGACCCTCGCCAGTGCTCTGCGCAGGATTGGAACATCAGTTA

90 ACATCTGACCACTGCCAGCCCACCCCCTCCCACCCACGTCC;ATTGCATCTCTGOGCTCCAGOGATAAAGCAGGTCTTOGGGTGCACC ATG ATT TCA CCA TTC TTA GTA CTG-17 Met Ile Ser Pro Phe Lou Val Lou

* * _ +* * * * * * *

201 GCC ATT GGC ACC TOC CTT ACT MC TCC TTA GTG CCA GAG AMA GAG AAM GAC CCC AAG TAC TOG CGA GAC CM GOG CM GAG ACA CTG AMA-9 Ala Ile Gly Thr Cys Lou Thr Asn Ser Lou Val Pro Glu Lys Glu Ly. Aso Pro Lys Tyr Try Ara Aso Gln Al. Gin Glu Thr Lou Lvs

* * * * * * * * *

291 TAT GCC CTG GAG CTT CAG AAG CTC MC ACC MC GTG GOCT AAG MT GTC ATC ATG TTC CTG GGA GAT GGG ATG GGT GTC TCC ACA GTG ACG+22 Tyr Al. Lou Glu Lou Gln Lys Lou Asn Thr Asn Va1 Ala. Lys Asn Va1 Ile Met Ph. Lou Gly Asp Gly Met Gly Val Ser Thr Vat Thr

381 GCT GCC CGC ATC CTC AAG GGT CAG CTC CAC CAC MC CCT GGG GAG GAG ACC AGG CTG GAG ATG GAC AAG TTC CCC TTC GTG GCC CTC TCC+52 Ala Als. Arg Ile Lou Lys Gly Gln Lou His His Asn Pro Gly Glu Glu Thr Are Lou Glu Met Asp Lys Phe Pro Phe Va1 Ala Leu Ser

* * * * * * * * *

471 AAG ACG TAC AAC ACC MT GCC CAG GTC CCT GAC AGT GCC G0C ACC GCC ACC GCC TAC CTG TGT GGG GTG AAG GCC MT GAG GGC ACC GTG+82 Lys Thr Tyr Asn Thr Asn Al& Gln Val Pro Asp S-r Al. Gly Thr Ala. Thr Al. Tyr Lou Cys Gly Val Lys Al. Asn Glu Gly Thr Va1

* * * * * * * * *

561 GGG GTA AGC GCA GCC ACT GAG CGT TCC COG TGC MC ACC ACC CAG CGG MC GAG GTC ACC TCC ATC CTG CGC TGG GCC MG GAC GCT GGG+112 Gly V.1 Ser Al. Al. Thr GCu Arg Ser Arg Cys I Thr Tb:I ln Gly Asn Giu Va1 Thr Sor Ile Lou Arg Trp Ala Lys Asp Ala Gly

* * * *----;--* * * * *

651 AM TCT GTG GGC AT? GTG ACC ACC AC AGA GCG M1C CAT CCC ACC CCC AGC GC GGCC TAC GOCC CAC TOG GOCT GAC CGG GAC TGG TAC TCA+142 Lyb Ser Va1 Gly Ile Va1 Thr Thr Thr Arg Va1 Asn His Al. Thr Pro Sor Al. Al. Tyr Ala His Sor Al. Asp Arg Asp Trp Tyr Ser

* * * * * * * * *

741 GAC MC GAG ATG CCC CCT GAG 0cc TTG AGC CAG GCC TOT AAG GAG ATC 0CC TAC CAG CTC ATG CAT MC ATC AGG GAC ATT GAC GTG ATC+172 Asp Asn Glu Mot Pro Pro Glu Al. Lou Sor Gln G1, Cys Lys Asp Ile Ala Tyr Gln Lou Met His Asn Ile Arg Asp Ile Asp Va1 Ile

* ********

831 ATG G0G GOT GGC COG AMA TAC ATO TAC CCC AMC MT AMA ACT GAT OTG 0OG TAT GAG AGT GAC GAG AAA GCC AGO GGC ACG AGG CTG GAC+202 Met Gly Gly Gly Arg Lys Tyr Met Tyr Pro LysAsn ys Tbr|Asp V.1 Gly Tyr Glu Ser Asp Giu Lys Ala Arg Gly Thr Arg Lou Asp

* * * * * * * *

921 GGC CTG GAC CTC GTT GAC ACC TOG AAG AGC TTC AMA COG AGA TAC AAG CAC TCC CAC TTC ATC TOG MC CGC ACG GAA CTC CTG ACC CTT+232 Gly Lou Asp Lou Va1 Asp Thr Trp Lys Ser Ph. Lys Pro Arg Tyr Lys His Sor His Ph. Ile Trp[Asn Arg Thr]Glu Lou Leu Thr Leu

* * * * * ***

1011 GAC CCC CAC MT GTG GAC TAC CTA TTG GOT CTC TTC GAG CCA 000 GAC ATO CAG TAC GAG CTG MAC AO MC AAC GTG ACG GAC CCG TCA+262 Asp Pro His Asn Va1 Asp Tyr Lou Lou Gly Lou Ph. Clu Pro Gly Asp Mot Gin Tyr Giu Lou Asn Ara As Asn Val Thr|Asp Pro Ser

* * * * * * * * *

1101 CTC TCC GAG ATO GTG GTG GTG GCC ATC CAG ATC CTC COG MG MC CCC AAM GGC TTC TTC TTG CTG GTG GM GGA GGC AGA ATT GAC CAC+292 Lou Ser Glu Mot Va1 Val Va1 Al. Il- Gln Ile Lou Arg Lys Asn Pro Lys Gly Ph. Ph. Lou Lou Val Glu Gly Gly Arg Ile Asp His

* * * * * * * * *

1191 G00 CAC CAT GM GGA AM GCC MG CAG 0CC CTGr CAT GAG 000 OTS GAG ATO GAc COG GCC ATC GOG CAG GCA GGC AGC TTG ACC TCC TCG+322 Gly His His Glu Gly Lys Ala Lys Gln Al. Lou His Glu Al. Val Glu Met Asp Arg Al. Ii- Gly Gln Ala Gly Ser Lou Thr Ser Ser

* * * * * * * * *

1281 GM GAC ACT CTG ACC GTG GTC ACT GC0 GAC CAT TCC CAGC TC TTC ACA TT? GOT GGA TAC ACC CCC CGT GGC MC TCT ATC TTT GGT CTG+352 Giu Asp Thr Lou Thr Val Va1 Thr Al. Asp His Sor His Va1 Ph. Thr Ph. Gly Gly Tyr Thr Pro Arg Gly Asn Ser Ile Phe Gly Lou

* * * * * * * * *

1371 GCC CCC ATG CTG AGT GAC ACA GAC MG MG CCC TTC ACT 0CC ATC CTG TAT GGC MT GGG CCT GGC TAC AMG GTG GTG GGC GGT GM CGA+382 Al. Pro Met Lou Ser Asp 7k Asp Lys Lys Pro Phe Thr AlaIl. Lou Tyr Gly Asn Gly Pro Gly Tyr Lys Val Val Gly Gly Glu Arg

* * * * * * * * *

1461 GAG AT GTC TCC ATG GTG GAC TAT OCT CAC MC MAC TAC CAG GCG CAG TOT 6CT GTG CCC CTG CGC CAC GAG ACC CAC GGC GGG GAG GAC+412 GluJ~sn Val SerjMet Va1 Asp Tyr Al. His Asn Asn Tyr Gln Ala Gln Ser Al. Val Pro Leu Arg His Glu Thr His Gly Giy Glu Asp

* * * * * * * * *

1551 GTG GCC GTC TTC TCC MA GGC CCC ATG GOG CAC CTG CTG CAC GGC GTC CAC GAG CAG MC TAC GTC CCC CAC GTG ATG GCG TAT GCA GCC+442 V.l Ala Val Phe Ser Lys Gly Pro Met Ala His Leu Lou His Gly V1l His Glu Gln Asn Tyr Val Pro His Val Met Ala Tyr Ala Ata

* * * * * * * * *

1641 TOGC ATC GGG GCC MC CTC GGC CAC TGT OCT CCT GCC AGC TCG GCA GGC AGC CTT GCT GCA GGC CCC CTG CTC GTC GCG CTG GCC CTC TAC+472 Cys Ile Glv Al. Asn Lou GlO His Cys Ala Pro Alt Ser Ser Aia Gly Ser Lou Al. Al. Gly Pro Lou Lou Va1 Ala Lou Ala Leu Tyr

* * * * * * * * * * *

1731 CCC CTG AGC GTC CTG TTC TGA GGGCCCAGGGCCCGGGCACCCACMGCCCGTGACAGATGCCMCTTCCCACACGGCAGCCCCCCCCTCAAGGGGCAGGGAGGTGGGGGCCT+502 Pro Lou Ser Va1 Lou Phe ---

* * * * * * * * * * * *

CCTCAGCCTCTGCAATCAAGAAGGGCCCAGACATCTGCCGCCCACCTCC CTCCCCTCTGGAATCTTCCCCAAGCCACCCACTTCTGCCTCCAGCCTTTGCTCC* * * * * * * * * * * *

CTCCCO(CTGCCCTTTGGCCACCACTAGATTTCTCTTGGGCAGCGAATACAGACTGC AGACATTCTC AAAGCCTCTTATTTTTCTAGCGAACGTATTTCTCCAGACCCAGAGG* * * * * * * * * * **CCCTGAAGCCTCCGTGGACATTGTGGATCTGACCCTCCCAGTCTCATCTCCTGACCCTCCCACTCCCATCTCCTTACCTCTGGACCCCCCAGGCCCTACAATGCTcATGTCCCTG;TC

* * * * * * * * * * * *CCCAGCCG*GCCCTCCTTCAGGGGA*TTGAGGTCTTTCTCCTCAGGACAGGCC*TTGCTCACTCACTCACTCCMGACCACCAGGGTCCCAGGAAGCCGGTGCCTGGGTGGCCATCCTA

CCCAGCCGTGCCCAGGCCCGGGUAGACCACCTGGCAGGGCTCACACTCCTGGGCTTTGACACACACAC CCAGCTCCTCTTTGAAGCGACTCTCCTGTTTGGAACGGCAAAAATTTT

TTTTTCTCTTTTTGGTGGTGGTTAAAAGGGAACAMCAAAACATTTAAATMAAACTTTCCAAATATTTC

FIG. 2. DNA sequence and deduced amino acid sequence of the L/B/K ALP cDNA. Numbers preceded by + or - refer to amino acidpositions. All other numbers refer to nucleotide positions. Asterisks occur at 10-base intervals. Amino acids -17 to -1 comprise a putative signalpeptide. A vertical line precedes amino acid + 1, the amino-terminal residue found in the mature protein. Amino acid residues that have beendetermined by protein sequence analysis of purified liver ALP are underlined. Five potential N-linked glycosylation signals, Asn-Xaa-Thr/Ser,are boxed. A 12-bp direct repeat in the 3' untranslated region of the cDNA is labeled by arrows. A single poly(A) addition signal AATAAA isunderlined twice.

contains 17 amino acids, mostly hydrophobic, at the amino extra residues, -17 to -1 in Fig. 2, presumably represent aterminus that are not present in mature liver ALP. These signal peptide.

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Proc. Natl. Acad. Sci. USA 83 (1986) 7185

DISCUSSIONALPs are present in most organisms with the exception ofsome plants (1)-. In higher organisms, the protein products ofmultiple ALP genes are expressed in specific tissues atvarious times during development (2). The phylogeny ofthese enzymes has been partially defined by comparison oftheir immunologic properties (2) and of amino-terminal poly-peptide sequences (23, 26). Analysis of cDNAs that encodethe different ALPs allows a more detailed investigation ofstructural and evolutionary relationships within this multi-gene enzyme family.The complete amino acid sequences of three ALP proteins

are now known. A computer-assisted comparison of E. coli(471 amino acids) (27, 42), human placental (type 1) (535amino acids) (9, 28), and human L/B/K ALP (524 aminoacids) precursor proteins is shown in Fig. 3. Amino acidpositions that are identical in all three proteins, or in the twohuman proteins, are boxed. Gaps have been introduced intothe protein sequences to maximize alignment of homologousregions.The alignment in Fig. 3 results in 52% amino acid identity

between the human ALPs (L/B/K and placental) over their

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A RA KA L

entire polypeptide lengths. At the DNA level, L/B/K andplacental ALP are 60%o homologous in the coding regions butno homology is detected between the cDNAs in the 5' and 3'untranslated regions (data not shown). As expected, there isless homology between the E. coli and mammalian ALPs.Thus, E. coli ALP is 25% homologous to L/B/K ALP and29% homologous to placental ALP over the 471 amino acidsof the E. coli enzyme.

Inspection of Fig. 3 reveals several areas that are highlyconserved in all three ALP polypeptides. These are the sameregions detected by Millan (28) and Kam et al. (29) in theircomparisons of placental and E. coli ALPs. These areasrepresent conservation of amino acids that comprise theactive site region in theE. colienzyme (30, 31). There are alsoseveral regions that are conserved only between the humanL/B/K and placental ALPs, presumably representing func-tions of mammalian ALPs not present in E. coli. TwoN-linked glycosylation signals at homologous sites occur inthe L/B/K and placental ALPs, though the L/B/K enzymecontains three additional glycosylation signals that are absentin placental ALP.As shown in Fig. 3, homology between the human placen-

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21 E22 P22 L71 E62 P63 L119 E111 P112 L161 E161 P162 L209 E206 P208 L259 E248 P253 L308 E297 P301 L358 E346 P350 L404 E395 P400 L425 E445 P450 L449 E495 P500 L513 P507 L

FIG. 3. Comparison of the amino acid sequences of E. coli (E), human placental (P), and human L/B/K (L) ALP precursor proteins. Gapsthat have been introduced into the sequences to maximize pairing of homologous amino acids are indicated by -. Amino acid + 1 correspondsto the first residue in each of the mature proteins. Amino acids that are identical in all three proteins or in the two human proteins are boxed.Amino acids that comprise the active site region in the E. coli ALP, including metal ligand sites (m) and residues that interact with substratephosphate (e), are indicated. Amino acids are shown in the single-letter code.

Biochemistry: Weiss et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

tal and L/B/K ALPs is decreased over the -50 carboxyl-terminal amino acids. However, each region contains astretch of hydrophobic residues that could participate inmembrane localization. These hydrophobic regions mayserve as membrane anchor domains that are present in themature ALPs, although another possibility exists. There isevidence to suggest that mammalian ALPs are attached to thecell surface membrane by a covalent linkage with phospha-tidyl inositol (32). Two other proteins with similar membraneattachments, thy-i and the trypanosome variable surfaceglycoprotein, contain carboxyl-terminal hydrophobic regionsthat are cleaved from the newly synthesized polypeptides(33-35). Similar proteolytic events may occur during theprocessing of the human ALPs. Amino acid sequence anal-ysis of liver ALP CNBr peptides confirms that amino acidsextending to at least Ser-484 are present in the matureL/B/K-type ALP. Precise definition of the carboxyl termi-nus of the mature ALPs awaits further studies on the purifiedproteins.Our data allow us to address the question of whether the

ALPs expressed in liver and bone are encoded by the samegene. We have used antiserum prepared against human liverALP to screen a Saos-2 cDNA expression library and isolatean immunoreactive recombinant phage that encodes a L/B/K-type ALP. Saos-2 is an osteosarcoma-derived cell line thathas been shown to produce relatively large amounts ofL/B/K-type ALP but no placental or intestinal ALP (11).Saos-2 cells are osteoblastic in nature, as demonstrated bytheir ability to produce cyclic AMP appropriately in responseto parathyroid hormone (7). This property has been shown tooccur in the line of Saos-2 cells that we have used to constructour cDNA library (Gideon Rodan, personal communication).We therefore believe that the Saos-2 ALP cDNA representsbone ALP. The polypeptide encoded by this cDNA isidentical to human liver ALP at the 87 amino acid positionsthat we have determined by sequencing the purified liverprotein. These data support the hypothesis that liver andbone ALPs contain the same protein moieties and thereforeare likely to be encoded by the same gene. This issue shouldbe resolved by using the Saos-2 ALP cDNA as a hybridiza-tion probe to isolate and analyze liver ALP cDNA andgenomic L/B/K ALP DNA.For many years, ALP has been thought to play a role in

bone mineralization, although the exact nature of this role isunknown (36). Bone ALP is an osteoblastic marker and theappearance of this enzyme coincides with bone formation invivo and in vitro (36). Evidence implicating ALP in bonemineralization is provided by the rare genetically determineddisease hypophosphatasia (37-39), which is characterized byabnormally low levels ofL/B/K ALP in all tissues, includingbone, but normal levels of placental and intestinal ALP (40,41). Patients with this disease suffer from defectiveosteogenesis. The most severe cases are lethal in infancy,with virtually complete absence of L/B/K ALP in all tissues(41). The genetic defects that produce hypophosphatasia areunknown. The cDNA probe for L/B/K ALP will be of use inelucidating this disease at the molecular level.We thank Ms. Joanne L. Borthwell for her assistance in the

preparation of the manuscript, Denise Kubaska for tissue culturework, Thomas Fischer for help with amino acid sequence analysis,and Dr. Mortimer Poncz for helpful advice and discussions. Thiswork was supported by National Institutes of Health Grant GM27018 and March of Dimes Grant 858. P.S.H. is supported byNational Institutes of Health Training Grant HD 07107 to theDivision of Biochemical Development and Molecular Diseases,

Children's Hospital ofPhiladelphia. M.J.W. is supported by NationalInstitutes of Health Medical Scientist Training Program Grant GM07170.

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7186 Biochemistry: Weiss et al.