Proc. Nati. Acad. Sci.

USA
Vol. 83, pp.' 7182-7186, October 1986
Biochemistry

Isolation and characterization of a cDNA encoding a human

liver/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 77225

Contributed by Harry Harris, June 10, 1986

tissues except that the bone isoenzyme has long been thought
to have a role in normal skeletal mineralization (6).
In this report, we describe the isolation and nucleotide
sequence analysis of a cDNA corresponding to a human
L/B/K ALP. This cDNA probably represents ALP of bone
origin since it was derived from Saos-2 osteosarcoma cells
that have osteoblastic properties (7). Partial amino acid
sequences obtained from purified liver ALP are identical to
portions of the polypeptide encoded by the L/B/K cDNA.
This is consistent with the hypothesis that the ALPs found in
liver and bone are identical at the amino acid level. Comparison of the protein sequences of human L/B/K, human
placental, and Escherichia coli ALPs shows conservation of
primary structure in catalytically important regions.

Alkaline phosphatases (ALPs) [orthophosABSTRACT

phoric-monoester phosphohydrolase (alkaline optimum), EC
3.1.3.1] isolated from human liver, bone, and kidney (L/B/K)
exhibit very similar biochemical and immunologic properties
that differentiate them from other human ALPs, such as those
characteristically found in placenta and intestine. Despite their
similarities, the L/B/K ALPs produced in different tissues
show slight physical differences. To examine structural and
evolutionary relationships between the various ALPs, a cDNA
corresponding to L/B/K ALP mRNA has been isolated. A
AgtlI cDNA expression library was constructed using poly(A)
RNA from the osteosarcoma cell line Saos-2 and screened with
anti-liver ALP antiserum. The 2553-base-pair cDNA contains
an open reading frame that encodes a 524 amino acid polypeptide with a predicted molecular mass of 57.2 kDa. This ALP
precursor protein contains a presumed signal peptide of 17
amino acids followed by 37 amino acids that are identical to the
amino-terminal sequence determined from purified liver ALP.
In addition, amino acid sequences of several CNBr peptides
obtained from liver ALP are found within the cDNA-encoded
protein. The deduced L/B/K ALP precursor polypeptide
shows 52% homology to human placental ALP and 25%
homology to Escherichia coli ALP precursor polypeptides.
Sixty percent nucleotide homology exists between the human
L/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 the
corresponding regions of placental ALP cDNA.

MATERIALS AND METHODS

Alkaline phosphatases (ALPs) [orthophosphoric-monoester

phosphohydrolase (alkaline optimum), EC 3.1.3.1] hydrolyze
various monophosphate esters at a high pH optimum. These
enzymes, which are widely distributed in nature, exist as
membrane-bound glycoproteins in higher organisms (1). The
enzyme products of at least three ALP gene loci [placental,
intestinal, and liver/bone/kidney (L/B/K)] are distinguishable in man by a variety of structural, biochemical, and
immunologic methods (2-5). The placental and intestinal
forms of human ALP are expressed in a relatively tissuespecific manner in normal individuals. In contrast, L/B/K
ALP is expressed at relatively high levels in osteoblasts and
to a lesser extent in fibroblasts, leukocytes, and cells from
numerous other tissues, including liver, kidney, breast, and
brain (1, 2). Slight differences in electrophoretic mobility and
thermostability between the L/B/K ALPs from various
tissues are attributed to differences in posttranslational
modification, although it is possible that their protein moieties are encoded by separate but related genes (2). Little is
known regarding the physiological function of ALPs in most

Purification of Liver ALP. Human liver ALP was purified

to homogeneity using an immunoaffinity column made with
monoclonal antibody ALPp/Sp2/19 as described (8).
Antiserum. Anti-human liver ALP antiserum was raised in
a rabbit using purified liver ALP as antigen.
Protein Sequence Determination. Amino acid sequence
analysis of purified liver ALP and its CNBr peptides was
performed as described (9).
Cell Lines. The Saos-2 cell line, originally obtained from
the laboratory of Jorgen Fogh (10), is derived from a human
osteogenic sarcoma. These cells have been shown to produce
relatively large amounts of ALP of the L/B/K type (11).
Isolation of Poly(A) RNA from Saos-2 Cells. Total cellular
RNA was isolated from Saos-2 cells using the guanidinium/cesium chloride method (12). Poly(A) RNA was isolated
by two rounds of oligo(dT)-cellulose chromatography (12).
cDNA Library Construction. Construction of a cDNA
library using the bacteriophage expression vector Xgtll was
carried 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 resultant library of 3 x 10' recombinant phage was amplified on E.
coli strain Y1088.
Screening cDNA Libraries with Antibody. The Saos-2
cDNA library was screened with rabbit anti-human liver ALP
antiserum according to Young and Davis (13). Bound antibody was detected using an avidin-biotin system (Vectastain
ABC Kit, Vector Laboratories, Burlingame, CA).
Nucleic Acid Hybridization Analysis. Radiolabeled DNA
probes were prepared either by nick-translation (Nick-Translation Kit, Bethesda Research Laboratories) or calf thymus

The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. 1734 solely to indicate this fact.

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.

7182

Biochemistry: Weiss et al.

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

DNA primer labeling (14). RNA was separated by electrophoresis 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 hybridized to 32P-radiolabeled probes according to the manufacturer's instructions. The membranes were washed under
stringent conditions (30 mM NaCl/3 mM sodium citrate for
1-2 hr at 680C) and autoradiographed.
Hybrid-Selected Translation. Twenty micrograms of cDNA
in plasmid vector pAT153 was linearized with EcoRI, denatured, and immobilized on a 0.6-cm square of nitrocellulose
paper (15). Hybridization to 15 ,ug of Saos-2 poly(A) RNA,
washing of the filter, and subsequent elution of RNA were as
described (12), except that RNA was eluted in a step-wise
fashion at 70, 80, and 1000C. The RNA was isolated by
ethanol precipitation and in vitro translation was performed
using a rabbit reticulocyte lysate system (Bethesda Research
Laboratories) with [35S]methionine according to the manufacturer's instructions. Immunoprecipitation ofin vitro translated protein was performed according to published procedures (16, 17). Polypeptides were fractionated on 10% polyacrylamide gels under denaturing conditions (18).
DNA Sequence Analysis. The 2553-base-pair (bp) L/B/K
ALP cDNA was sequenced by the Sanger dideoxy chaintermination method (19). Overlapping M13 subclones were
generated by using the T4 polymerase deletion method of
Dale et al. (20) and by subcloning various restriction fragments into M13. Both strands of the cDNA were sequenced
in full. Computer analyses of DNA and protein sequences
were performed using the IBI/Pustell DNA and Protein
Sequence Analysis System (International Biotechnologies,
New Haven, CT), FASTP (21), and NUCALN (22).

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

pS3-1

7183

cipitates is shown in Fig. 1A. A single 58-kDa band is

precipitated by anti-liver ALP and not by preimmune serum.
The size of this polypeptide is consistent with the expected
size of unglycosylated L/B/K ALP precursor protein (23).
Isolation of a L/B/K ALP cDNA from Saos-2 cDNA. The
Saos-2 cDNA library was screened with anti-liver ALP
antiserum. Seven immunoreactive clones were isolated from
screening 5 x 105 recombinant phage. The cDNA inserts
were subcloned into pAT153 and examined by DNA blot
hybridization analysis using the various candidate cDNA
inserts as hybridization probes. Four of the clones were of
identical size [2.5 kilobase pairs (kb)], hybridized to each
other, and reacted most intensely with the antiserum. A
hybrid-selection translation assay was performed to verify that
these 2.5-kb cDNAs encode ALP. A plasmid subclone of one
of the 2.5-kb cDNA inserts, pS3-1, was used to select crosshybridizing nucleic acid from Saos-2 poly(A) RNA. Fig. 1 B and
C show that the RNA selected by pS3-1 programed the
synthesis of a 58-kDa protein that was immunoprecipitable by
liver ALP antiserum. RNA blot analysis reveals that pS3-1
hybridizes to a single 2.6-kb band in Saos-2 RNA (data not
shown), 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 sequence
contains an open reading frame, beginning at nucleotide 177,
that encodes a 524 amino acid polypeptide. Nucleotides
surrounding the AUG initiation codon agree at all but the last
position with a consensus sequence, ACCAUGG, that has
been 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 by
immunoprecipitation of in vitro translation products directed
by Saos-2 RNA.
We and others (23, 26) have determined the sequence of 37
amino acid residues from the amino terminus of purified liver
ALP. An additional 50 residues of liver ALP were determined
from four CNBr peptides. These sequences of liver ALP (37
amino-terminal residues and 50 internal CNBr residues) are
identical to segments of the polypeptide sequence encoded by
the pS3-1 cDNA insert and are underlined in Fig. 2.
The polypeptide encoded by the L/B/K ALP cDNA
pAT 153
N

Nre'
A

4,zD
0tt
Nt

Sj

4;;,. IQI.'

9769-

97

69

97

-69
-

-46

46-

-430
-30
FIG. 1. (A) Immunoprecipitation of Saos-2 RNA in vitro translation products. Saos-2 poly(A) RNA was translated in a rabbit reticulocyte
lysate in vitro translation system, immunoprecipitated with either rabbit anti-liver ALP antiserum (aLALP) or preimmune serum, and
fractionated 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 in
vitro translation products of Saos-2 RNA that had been hybrid-selected by pS3-1 or pAT153 were immunoprecipitated by anti-liver ALP
antiserum and fractionated by NaDodSO4/PAGE. Molecular masses are shown in kDa.

7184

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

Biochemistry: Weiss et al.

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
+112 Gly V.1 Ser Al. Al. Thr GCu Arg Ser Arg Cys I Thr Tb:I ln Gly Asn
*
*
*
*----;--*
651 AM TCT GTG GGC AT? GTG ACC ACC AC AGA GCG M1C CAT CCC ACC CCC AGC
+142 Lyb Ser Va1 Gly Ile Va1 Thr Thr Thr Arg Va1 Asn His Al. Thr Pro Sor
*

GAG GTC ACC TCC ATC CTG CGC TGG GCC MG GAC GCT GGG
Giu Va1 Thr Sor Ile Lou Arg Trp Ala Lys Asp Ala Gly
*

GC GGCC TAC GOCC CAC TOG GOCT GAC CGG GAC TGG TAC TCA
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
ys Tbr|Asp V.1 Gly Tyr Glu Ser Asp Giu Lys Ala Arg Gly Thr Arg Lou Asp
+202 Met Gly Gly Gly Arg Lys Tyr Met Tyr Pro
*

LysAsn

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 --*

1843 CCTCAGCCTCTGCAATCAAGAAGGGCCCAGACATCTGCCGCCCACCTCC
*

CTCCCCTCTGGAATCTTCCCCAAGCCACCCACTTCTGCCTCCAGCCTTTGCTCC
*

1962 CTCCCO(CTGCCCTTTGGCCACCACTAGATTTCTCTTGGGCAGCGAATACAGACTGC AGACATTCTC AAAGCCTCTTATTTTTCTAGCGAACGTATTTCTCCAGACCCAGAGG

*
*
**
*
*
*
*
*
* CTCATCTCCTGACCCTCCCACTCC
*
*
2081 CCCTGAAGCCTCCGTGGACATTGTGGAT
CTGACCCTCCCAGT
CATCTCCT
TACC TCTGGAC
CCCCCAGGCC
CTACAATGCT
cATGTCCCTG;TC
*
*
*
*
*
*
*
*
*
*
*
*
2200 CCCAGCCG*GCCCTCCTTCAGGGGA*TTGAGGTCTTTCTCCTCAGGACAGGCC*TTGCTCACTCACTCACTCC
MGACCACCAGGGTCCCAGGAAGCCGGTGCCTGGGTGGCCATCCTA

2319 CCCAGCCGTGCCCAGGCCCGGGUAGACCACCTGGCAGGGCTCACACTCCTGGGCTTTGACACACACAC CCAGCTCCTCTTTGAAGCGACTCTCCTGTTTGGAACGGCAAAAATTTT

2438 TTTTTCTCTTTTTGGTGGTGGTTAAAAGGGAACAMCAAAACATTTAAATMAAACTTTCCAAATATTTC

FIG. 2. DNA sequence and deduced amino acid sequence of the L/B/K ALP cDNA. Numbers preceded by + or refer to amino acid
positions. All other numbers refer to nucleotide positions. Asterisks occur at 10-base intervals. Amino acids -17 to -1 comprise a putative signal
peptide. A vertical line precedes amino acid + 1, the amino-terminal residue found in the mature protein. Amino acid residues that have been
determined 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 is
underlined twice.
-

contains 17 amino acids, mostly hydrophobic, at the amino

terminus that are not present in mature liver ALP. These

extra residues, -17 to -1 in Fig. 2, presumably represent

signal peptide.

Biochemistry: Weiss et al.

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

DISCUSSION

entire polypeptide lengths. At the DNA level, L/B/K and

placental ALP are 60%o homologous in the coding regions but
no homology is detected between the cDNAs in the 5' and 3'
untranslated regions (data not shown). As expected, there is
less homology between the E. coli and mammalian ALPs.
Thus, E. coli ALP is 25% homologous to L/B/K ALP and
29% homologous to placental ALP over the 471 amino acids
of the E. coli enzyme.
Inspection of Fig. 3 reveals several areas that are highly
conserved in all three ALP polypeptides. These are the same
regions detected by Millan (28) and Kam et al. (29) in their
comparisons of placental and E. coli ALPs. These areas
represent conservation of amino acids that comprise the
active site region in theE. coli enzyme (30, 31). There are also
several regions that are conserved only between the human
L/B/K and placental ALPs, presumably representing functions of mammalian ALPs not present in E. coli. Two
N-linked glycosylation signals at homologous sites occur in
the L/B/K and placental ALPs, though the L/B/K enzyme
contains three additional glycosylation signals that are absent
in placental ALP.
As shown in Fig. 3, homology between the human placen-

ALPs are present in most organisms with the exception of

some plants (1)-. In higher organisms, the protein products of
multiple ALP genes are expressed in specific tissues at
various times during development (2). The phylogeny of
these enzymes has been partially defined by comparison of
their immunologic properties (2) and of amino-terminal polypeptide sequences (23, 26). Analysis of cDNAs that encode
the different ALPs allows a more detailed investigation of
structural and evolutionary relationships within this multigene 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) (535
amino acids) (9, 28), and human L/B/K ALP (524 amino
acids) precursor proteins is shown in Fig. 3. Amino acid
positions that are identical in all three proteins, or in the two
human proteins, are boxed. Gaps have been introduced into
the protein sequences to maximize alignment of homologous
regions.
The alignment in Fig. 3 results in 52% amino acid identity
between the human ALPs (L/B/K and placental) over their

-22
-2 2

QST

M K

LALLPL FTPVTKAR

LLILIG

L R LQ

IJI

SP F V
G D QfA A L R D SL S D K
R
- -.
K LQP A Q T A E L K LNTN V

-1 7

AR
AK
AL
F K G ID
ET

PLJ

RL

A K N L I

NK

RFPYVA

VI

E MP V L

N R A

V D

NT

N A Q

V P

G P

V T S R K

D S
F

A G

Q K C P G N A;;L K

A T

G K

rCTT

T A Y

L L

DA D VPASAR Q EGCQDIAT
K I YLQ
UJSA DLBIDIN E MW P ELAJL S Q
IHITV NIRINIW

- - - -S

T F A E T A T A G E W Q G K T L R E OA Q A R
T R L D
;;1FRMGT PE PE P D Y S QGG
Y P K N K T V G LSE S E K A RLG T R L D
A D G N M P V R
N R T E |MQ A
N R T E L --

S|L D

K A.T

EY Q|L V|S
Gl K N IL VlV Gl L D 3L -

Y H G ,N I D K

IVT H
H NID YW

A P G G

AWSTGVK-T Y N G

HIV PD SIGAITATAYL CG VKIG

- - -E K D H P T I L E M AK A
N
D H - - - A R F N Q C N T T|R|G N E V I V M N R|A K|K|A G K S V G|V|VT T T
I
E VTV
I L R
D GK S V G
AT E R S R

L
L
Vl

Q G D I

LL G D G M D
AAR N Y A E G A G G F
IFL G D G M G V S T V T A A R I L K G Q K K D K
M F L G D GMG V S T V T A A R I L K G Q L H H N

PDY

KTC

,S K TYN

A T. K T Y

PA K N I I

AR P L T G Q Y T H

EI PLAM-

LflP

-_- - - IT

S LG I I V EIE NIPID FIN R EAIA- --EAMG A

A I G T C T N S L V E K K D K Y RD Q Q
T KY

M L G P CML L L

D MIQI

FiQ

iI

E G

JV

T.

CG

QDT RA A L
V|Q| A|S|P|A G T|YA

V K

IH

AR

AITS

A AI

RL

I SINIM -IDIDVIILIG G GRKY

D V IM
R K Y
LIM HJI R

D A A S L N
- - - - Q E
- - - - D T

1 C T P N P

MG L F E P G D MIK

LI

SV T E A N Q Q K P L L G L F
Wi L S F K AP KR YR QK GH AS RH FY VI
K
- - -

R N D S

D K

A I

I HIRiD S T LID PSLMIE MIT E AIAIL R

L NN N V T
V VJI Q

P S LIS! MV

AN P C G
I GfT V D Lf
LflS K WEK G F F L
E OVQ RflE FLALK,
|QQGIj )I
S R
LILIS R N PRG F F L PV E G G R I D H G H
RATIEIT I M F|D|D A IE RIA GIQI T -S
KAKQQ
S
IJ R K
G Q
S L T
PI
L
E
RGK
LI
HJA V E M R

V I VT A D
L|V TA D

E G N

TL S

S Q I
ISF

H S HV

D T K
SS

V A - - - P
Y P LE
G

A L N T K1 G A V M V M
RIDR A Y TV LY

I F G LAPG K- A

G NI

NS I F G L FDA
A
L S D
P
TTK
D TLT PTM
VIV T TA
DT
IT PI
I|L Y G N

_
IG PG YV L
-

G PG

- - - - - - - - -

K V V G

P D

EE
JDT

NN

L F

S E E D S Q E -

T E S E S G S P E
Y

M A L
F A
VMA

IAHLVGV|Q IE QI T F IAfH
N Y V P
H
|AHLT LL
L LP L LAGT L L L L ETATAP
YP LSVLF
T

- - - -

- - - - - -

R Q Q S A V P L D E

S M V D Y A H N NWQ A

s A v P

LR

--

H
T G S Q L R I A A Y G
HA G E D V A V F A R G P Q

HE TGG E D V A

L K
L E P YTAC D LAP P AG T T D
AAC I GAN LG H CAP AS SAG S L
G

7185

F|S

PIM

H P G R S V V P A
G P L L VAL A L

21

22 P
22 L
71

62
63
119
111
112

P
L
E
P
L

161 E
161 P

162
209
206
208
259
248
253
308
297
301
358
346
350
404
395
400
425
445
450
449
495
500

L
E
P
L
E
P
L
E
P
L
E
P
L
E
P
L
E
P
L
E
P

513 P

507 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. Gaps
that have been introduced into the sequences to maximize pairing of homologous amino acids are indicated by -. Amino acid + 1 corresponds
to 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 substrate
phosphate (e), are indicated. Amino acids are shown in the single-letter code.

7186

Biochemistry: Weiss et al.

tal and L/B/K ALPs is decreased over the -50 carboxylterminal amino acids. However, each region contains a
stretch of hydrophobic residues that could participate in
membrane localization. These hydrophobic regions may
serve as membrane anchor domains that are present in the
mature ALPs, although another possibility exists. There is
evidence to suggest that mammalian ALPs are attached to the
cell surface membrane by a covalent linkage with phosphatidyl inositol (32). Two other proteins with similar membrane
attachments, thy-i and the trypanosome variable surface
glycoprotein, contain carboxyl-terminal hydrophobic regions
that are cleaved from the newly synthesized polypeptides
(33-35). Similar proteolytic events may occur during the
processing of the human ALPs. Amino acid sequence analysis of liver ALP CNBr peptides confirms that amino acids
extending to at least Ser-484 are present in the mature
L/B/K-type ALP. Precise definition of the carboxyl terminus of the mature ALPs awaits further studies on the purified
proteins.
Our data allow us to address the question of whether the
ALPs expressed in liver and bone are encoded by the same
gene. We have used antiserum prepared against human liver
ALP to screen a Saos-2 cDNA expression library and isolate
an immunoreactive recombinant phage that encodes a L/B/
K-type ALP. Saos-2 is an osteosarcoma-derived cell line that
has been shown to produce relatively large amounts of
L/B/K-type ALP but no placental or intestinal ALP (11).
Saos-2 cells are osteoblastic in nature, as demonstrated by
their ability to produce cyclic AMP appropriately in response
to parathyroid hormone (7). This property has been shown to
occur in the line of Saos-2 cells that we have used to construct
our cDNA library (Gideon Rodan, personal communication).
We therefore believe that the Saos-2 ALP cDNA represents
bone ALP. The polypeptide encoded by this cDNA is
identical to human liver ALP at the 87 amino acid positions
that we have determined by sequencing the purified liver
protein. These data support the hypothesis that liver and
bone ALPs contain the same protein moieties and therefore
are likely to be encoded by the same gene. This issue should
be resolved by using the Saos-2 ALP cDNA as a hybridization probe to isolate and analyze liver ALP cDNA and
genomic 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 is
unknown (36). Bone ALP is an osteoblastic marker and the
appearance of this enzyme coincides with bone formation in
vivo and in vitro (36). Evidence implicating ALP in bone
mineralization is provided by the rare genetically determined
disease hypophosphatasia (37-39), which is characterized by
abnormally low levels of L/B/K ALP in all tissues, including
bone, but normal levels of placental and intestinal ALP (40,
41). Patients with this disease suffer from defective
osteogenesis. 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 are
unknown. The cDNA probe for L/B/K ALP will be of use in
elucidating 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 culture
work, Thomas Fischer for help with amino acid sequence analysis,
and Dr. Mortimer Poncz for helpful advice and discussions. This
work was supported by National Institutes of Health Grant GM
27018 and March of Dimes Grant 858. P.S.H. is supported by
National Institutes of Health Training Grant HD 07107 to the
Division of Biochemical Development and Molecular Diseases,

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

Children's Hospital of Philadelphia. M.J.W. is supported by National
Institutes of Health Medical Scientist Training Program Grant GM
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