0013-7227/92/1304-1957$03.oo/O Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 130,No. 4 Printed

Ontogeny, Immunocytochemical Localization, and Biochemical Properties of the Pregnancy-Associated Uterine Elastase/Cathepsin-G Protease Inhibitor, Antileukoproteinase (ALP): Monospecific Antibodies Synthetic Peptide Recognize Native ALP* ROSALIA C. M. SIMMEN, FRANK J. MICHEL, ALBERT LESLIE C. SMITH, AND MARIA FILOMENA VENTURA Department

of

Animal

Science, University

of

Florida,

Gainesville,

Florida

Expression of the mRNA encoding the elastase/ ABSTRACT. cathepsin-G protease inhibitor, antileukoproteinase (ALP), is highest in pig uterus during mid- and late pregnancy, suggesting a stage of pregnancy-dependent role for ALP in feto-maternal interactions. To elucidate a function for ALP in these events, immunogenic probes were developed to localize sites of ALP expression in the environment of the developing fetus. Monospecific antibodies raised against a 16-mer synthetic peptide corresponding to residues 21-36 (ALP 16P) of the deduced amino acid sequence of pig uterine ALP were generated by active immunization of sheep. ALP 16P conjugated to keyhole limpet hemocyanin elicited high titer antibodies that were specific to ALP. The antipeptide antibodies were used to characterize pig uterine ALP from allantoic fluids. Uterine ALP has an approximate mol wt of 14,000 and a p1 of 8.2 and exhibits elastase inhibitor activity. Amino-terminal amino acid sequencing of uterine ALP indicated the sequence ABNALKGGACPPRKIVQC, which has 44% identity with the corresponding region in

M

of the placental-uterine interAINTENANCE phase and integrity of the placenta are essential for successful progression of pregnancy and normal fetal development. Potentially important roles for proteases and protease inhibitors in these processes have been suggested (1,2); however, only limited data are available describing their respective sites and mechanisms of action. We have recently isolated and characterized cDNA clones for the mRNA encoding a pregnancy-associated pig uterine protease inhibitor, antileukoproteinase (ALP) (3). ALP is a low mol wt (M,) protein whose major substrates in uiuo are elastase and cathepsin-G, enzymes

in U.S.A.

to a

E. FLISS, FLISS 32611

human bronchial ALP. RIA for ALP, developed using ALP 16P as standard and iodinated tracer, demonstrated the presence of immunoreactive ALP in early, mid-, and late pregnant endometrium and myometrium, placenta, allantoic fluids, fetal cord blood, and fetal liver. ALP was undetectable in the maternal circulation. The ALP levels in endometrium, allantoic fluids, and fetal cord blood changed with the stage of pregnancy; however, ALP content in placenta, myometrium, and fetal liver, although different among tissues, remained invariant during gestation. By immunocytochemical analyses, ALP was localized in the glandular epithelium of the uterus, in placenta, and in fetal liver, consistent with the presence of immunoreactive ALP as measured by RIA. The localization of uterine ALP in placenta and its corresponding transport to fetal circulation provide strong evidence to support a physiological function for the protease inhibitor in the biological mechanisms controlling fetal development in utero. (Endocrinology 130: 1957-19651992)

that degrade structural components of cell membranes and microcapillary network adjacent to mucosal regions (4, 5). ALP has been localized in the human cervix (6) and adult and fetal human lung (7, 8) and has been purified from the parotid and submandibular glands, saliva, tears, and gut (9,lO). However, the demonstration of synthesis of ALP by the pregnant pig uterus was the first in any mammalian uterus to date (3). The expression of ALP mRNA in pig uterus was greater than that in any maternal or fetal tissue examined, including lung, was pregnancy stage dependent, and was differentially regulated by exogenous estrogen and progesterone (3, 11). These data suggested that ALP may have a function in pregnancy related to fetal development. To clarify the role of ALP in uiuo during pregnancy, we have produced and characterized antiserum against a synthetic peptide corresponding to residues 21-36 of the deduced amino acid sequence of porcine ALP. This anti-

Received October 14,1991. Address all correspondence and requests for reprints to: Dr. Rosalia C. M. Simmen, Department of Animal Science, 125 Animal Science Building, University of Florida, Gainesville, Florida 32611. * This work was supported in part by NIH Grant HD-21961 and USDA Grant 91-372025-6333. This manuscript is published as Journal Series R-03135, University of Florida, Agricultural Experiment Station. 1957

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ANTIPEPTIDE

1958

ANTIBODIES

serum was used to characterize uterine ALP, to quantify levels of this protein in various biological fluids and reproductive tissues, and to elucidate potential sites of ALP action during pregnancy in the pig.

Materials

and Methods

Preparation of synthetic peptide and antisera The antigenic segment corresponding to the region of highest hydrophilicity was predicted from analysis of the deduced amino acid sequence of porcine ALP (3), following previously described computer programs (12,13) included in the sequence analysis software package of the Genetics Computer Group (University of Wisconsin, Madison, WI). Based on this analysis, the peptide corresponding to amino acid residues 21-36 (YEKPKCTSDWQCPDKK) was synthesized using an Applied Biosystem 43OA peptide synthesizer (Applied Biosystems, Foster City, CA). The synthetic peptide (ALP 16P) was analyzed for purity (>90%) by reverse phase HPLC and for amino acid composition before use for antibody production. Antibodies to ALP 16P were raised in sheep by active immunization. ALP 16P was conjugated to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO) with m-maleimidobenzoyl-iv-hydroxysuccinimide ester (Pierce, Rockford, IL) following previously described protocols (14). The conjugated peptide (4 mg in 0.5 ml saline solution) was mixed with an equal volume of Freund’s complete adjuvant, and the resultant emulsion was injected im in sheep. Subsequent injections using the same dose of conjugated peptide in incomplete Freund’s adjuvant were given every other week for the next 4 weeks, at which time serum was tested for antibody titer by RIA (see below). Booster injections were given at monthly intervals, and blood was collected approximately 10 days after each boost. The antiserum collected after the eighth boost was used for all studies described in this paper. Antibody titer determination The titer of the antipeptide serum was determined by incubating equal volumes (100 ~1) of various dilutions of the antiserum with ‘261-labeled ALP 16P in 100 mM phosphate buffer (pH 7.0), 1 mM EDTA, 0.5% Tween-20, and 0.1% gelatin at 4 C overnight. ALP 16P was radiolabeled with Na1251 by the Iodogen method (15) to a specific activity of 38.2 &i/pg. The antibody-peptide complex was precipitated by the addition of 200 ~1 rabbit antisheep immunoglobulin (IgG) previously diluted 1:32 in 100 mM phosphate buffer, pH 7.0; incubation for 2 h at room temperature; and addition of polyethylene glycol to a final concentration of 6%. The samples were centrifuged at 4000 x g for 20 min, and pellet-bound radioactivity was counted in a Cobra autogamma counter (Packard Instruments, Downers Grove, IL). RIA procedure Immunoreactive antigen in pig tissue cytosolic extracts and biological fluids was quantitated by RIA. Tissue cytosolic extracts were prepared as described previously (16). All assays were carried out in triplicate in 12 x 75-mm glass tubes, following the procedure described above in a total assay volume

TO UTERINE

ALP

Endo. 1992 Voll30. No 4

of 1 ml. [iZI]ALP 16P, diluted in assay buffer to 30,000 cpm (1.8 ng)/lOO ~1, was added to each assay tube. Unlabeled peptide was used at doses ranging from 0.078-50 ng to generate a standard curve. The sensitivity of the assay was 0.15 rig/tube. Nonspecific binding was typically l-2% of the labeled peptide added. The intra- and interassay coefficients of variation were 2.2% and 16.9%, respectively. Purification

of antipeptide serum IgG fraction

Serum IgG was purified by chromatography on DEAE-Cibacron blue agarose (Sigma) in a bed volume of 7 ml/ml serum. The column was preequilibrated with 2 ~010.02 M K2HP04 (pH S.O)-0.2% NaN3 before loading of serum, which was previously dialyzed in the same buffer. The IgG fraction was eluted with 2 vol equilibration buffer, pooled, and subsequently used for immunoblotting. Analysis of this fraction on an sodium dodecyl sulfate (SDS)-polyacrylamide gel, followed by Coomassie blue staining demonstrated only the 45K and 22K M, bands, representing the heavy and light chains of IgG protein, respectively (data not shown). Electrophoresis and Western blot analysis Samples for immunoblot analysis were subjected to one- or two-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE), according to protocols described by Laemmli (17) and Roberts et al. (18). The two-dimensional SDS-PAGE for basic proteins used nonequilibrium pH gradient electrophoresis (NEPHGE). Proteins in gels were electrophoretically transferred to nitrocellulose filters (Schleicher and Schuell, Keene, NH), following previously described protocols (19). After the transfer, lanes containing M, markers were stained with Fast Green (0.1% in methanol-acetic acid; Sigma). The nitrocellulose sheet was rinsed twice with Tris-buffered saline (10 mM Tris and 160 mM NaCl, pH 7.4; TBS) containing 0.1% Tween-20, incubated overnight with 1% Blotto (dry fat milk) in TBS, and then incubated with sheep antipeptide antiserum (I:500 dilution) in TBS and 1% Blotto. After a 2-h incubation at room temperature with gentle shaking, the membrane was rinsed twice with TBS-Tween and incubated with an alkaline phosphatase conjugate of rabbit antisheep IgG, diluted l:lO,OOO in TBS and 1% Blotto. The membrane was washed twice with TBS and then incubated for 2-5 min with substrate buffer (100 mM Tris, 100 mM NaCl, and 50 mM MgCl,, pH 9.5). Protein bands were visualized by incubating the membranes sequentially with nitro blue tetrazolin (50 mg/ml in dimethylformamide) and 5-bromo-4-chloro-3-indoyl phosphate, diluted I:15 and 1:30, respectively, in 100 mM Tris (pH 9.5), 100 mM NaCl, and 5 mM MgC12. The reaction was allowed to proceed until the desired color intensity was achieved and then stopped by rinsing the membrane in deionized water. Immunocytochemical

studies

Tissues were fixed in Bouin’s reagent and embedded in paraffin (20). Preparation of tissue sections before incubation with primary antibody was performed as previously described (21). The IgG fraction of ALP antiserum was diluted 1:lO in 10 mM PBS (pH 7.4) and applied to tissue sections (5 pm) mounted on glass slides. As negative controls, adjacent tissue sections

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ANTIPEPTIDE

ANTIBODIES

were incubated with purified IgG fraction of preimmune sheep serum at the same dilution as the test antibody. Slides were incubated overnight in a humidified chamber at room temperature and subjected to the avidin-biotin staining procedure, described in the immunohistochemical staining kit (Biomeda, Foster City, CA). The tissues analyzed were taken from animals on days 60 (endometrium and placenta) and 75 (fetal liver) of pregnancy, respectively. Analysis of ALP in allantoic fluids (AF) AF from day 75 pregnant gilts were collected during surgery, as described previously (22). The fluids were clarified by low speed centrifugation, and proteins were precipitated with ammonium sulfate (to 80% final concentration) at 4 C for 1 h. The pellet was recovered by centrifugation (48,500 X g) for 20 min in an SS34 (Beckman, Palo Alto, CA) rotor and dissolved in 10 mM Tris-HCl, pH 8.2, to a final volume of 32 ml/500 ml starting volume of AF. Samples (16 ml corresponding to -200 mg total protein) were then applied to a Sephadex G-100 gel filtration column (total bed volume, 500 ml) equilibrated with 10 mM Tris, pH 8.2, and proteins were eluted in the same buffer at a flow rate of 40 ml/h. Fractions (7 ml/tube) were collected and assayed for the presence of ALP by RIA. Peak immunoreactive fractions were pooled, assayed for elastase inhibitor activity (see below), and analyzed by one- and twodimensional SDS-PAGE, followed by immunoblotting. The Sephadex G-100 column was calibrated by elution of M, standards comprising a mixture of bovine y-globulin (M,, 158,000), chicken ovalbumin (M,, 44,000), horse myoglobin (M,, 17,000), and bovine insulin (M,, 5,700).

TO UTERINE

ALP

1959

destained in 50% methanol. The protein spot corresponding to ALP, previously identified by immunoblot analysis of an identical gel, was sequenced using an Applied Biosystems model 470A Gas Phase Protein Sequencer with an on-line analytical HPLC system. Statistical analyses The statistical significance of RIA results from biological samples at different stages of pregnancy was determined by least squares analyses of variance, using the General Linear Models procedure of the Statistical Analysis System (SAS) (24). In all cases, P < 0.05 was considered significant.

Results Characterization

of anti-ALP

peptide polyclonal antibody

A 16-mer synthetic peptide representing the region of highest hydrophilicity in the deduced amino acid sequence of porcine ALP was conjugated to KLH and injected im in sheep. This peptide was antigenic and

resulted in the production of antibodies after the eighth boost. The titer of the antibody was determined by using increasing centration

dilutions of the antiserum with a fixed conof [‘251]ALP 16P to estimate the amount of labeled peptide bound. The results presented in Fig. 1

show a linear decrease in the amount of labeled peptide bound with increasing dilutions of the antiserum. Using a 1:8000 dilution of the primary antiserum and a 1:150 dilution of the secondary antiserum (rabbit antisheep),

Elastase inhibitor activity

approximately 50% of [‘251]ALP 16P was bound. The binding of the antibody to the labeled antigen was spe-

The protease inhibitor activity of uterine ALP was measured by assaying residual activity of elastase toward a synthetic substrate after coincubation of elastase and ALP (23). Partially purified ALP (from Sephadex G-100 column chromatography of day 75 AF) was incubated with pig pancreatic elastase (final concentration, 0.16 PM; Sigma) in 0.1 M HEPES buffer, pH 7.5, containing 1 M NaCl for 30 min at room temperature. The elastase substrate Suc-(Ala)a-pNA (Sigma) dissolved in N,Ndimethylformamide was then added to the reaction at a final concentration of 0.4 nM. After 30 min at room temperature, the reaction was stopped by the addition of 30 ~1 glacial acetic acid, and absorbance at 410 nM was measured. A standard curve was generated using increasing concentrations of elastase (0.032-0.32 pM) in the absence of coincubation with ALP. Inhibitory activity was expressed as a percentage of residual elastase activity, with the activity of added elastase in the absence of immunoreactive ALP being 100%.

cifically inhibited by the homologous peptide in a dosedependent manner (Fig. 2A). The lowest limit of detection was approximately 0.062 fmol/tube. Unrelated pep‘O0I D 5 mo %

60 --

2

40 --

bp

20 --

I

l\ .l.,

.I.

c-

04 0

NH2-Terminal amino acid sequences To purify ALP for amino-terminal amino acid sequencing, proteins from the G-100 fraction containing immunoreactive antigen were separated by two-dimensional SDS-PAGE using the NEPHGE system and electrophoretically transferred onto Immobilon PVDF membranes (Millipore, Bedford, MA) by the method of Towbin (19). The membrane was rinsed in water, stained with 0.01% Coomassie blue in 50% methanol, and

a0 --

1:2000

1:4000

1:6000

1:8000

l:lO.OOO

1:12,000

Ab dilution

1. Antibody (Ab) titer for RIA analysis.Equal volumes (100 al) of various dilutions of antiserum from sheep injected with KLHconjugatedsynthetic ALP peptide were incubated with [lz51]ALP16P. The antibody-bound labeled peptide was precipitated with rabbit sntisheepIgG. The x- and y-axesrepresentantibody dilution and labeled peptide bound asa percentageof the labeledpeptide added,respectively. Results are from triplicate determinations from two separate experiments. FIG.

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ANTIPEPTIDE

1960

-0

80--

00--

‘* ‘.

60 --

r\

20 --

o-o

UF

A-A A-A 0-0

Chymotrypsin Alpha-l antlttypsln ALP 16P

9

l\

40 --

ANTIBODIES

0

\

l

\

0

0 lo-2

1

16

Concentration

10-l

1

ALP 16 P (ng)

10’

lo2

102

103

(ng)

10’

or

VOLUME

(ul)

FIG. 2. Top panel, Specificity of binding of [‘*‘I]ALP 16P to anti-ALP antibody. [‘“I]ALP 16P and increasing concentrations of unlabeled ALP 16P, UP, chymotrypsin, and a+ntitrypain were incubated with antipeptide antibody (1:6000 dilution) under the conditions described in Moteriols and Methods. Percent B/B0 represents the ratio of specific binding of labeled ALP 16P with the antibody in the presence and absence of increasing amounts of unlabeled peptide or proteins. Bottom panel, Displacement of [1161]ALP 16P binding to anti-ALP peptide antibody by pig uterine and placental extracts. Increasing volumes (microliters) of cytosolic extracts of uterus and placenta from a day 60 pregnant pig were incubated with labeled ALP 16P as described above. Data are representative of two experiments, with each point representing the mean of triplicate determinations.

tides, including the porcine uterine iron-binding protein uteroferrin (UF) (25) and the protease inhibitors alantitrypsin and chymotrypsin (Sigma), did not compete for binding to the antibody, even in very high amounts. Cytosolic extracts prepared from day 60 pregnant pig uterus and day 60 pregnant pig placenta exhibited dilution curves parallel with that of the unlabeled peptide (Fig. 2B). Characteristics

of pig uterine ALP

AF from a midpregnant (day 75) pig, which exhibited high levels of ALP (see Table 2), was used as starting

TO

UTERINE

ALP

Endo. 1992 Vol13O*No4

material to partially purify ALP. AF proteins were precipitated with ammonium sulfate, and the precipitate was subjected to Sephadex G-100 gel filtration chromatography. A representative elution profile of immunoreactive ALP is shown in Fig. 3 (top panel). Fractions corresponding to peak immunoreactivity (tubes 38-41) were pooled and analysed by one (Fig. 3A, bottompanel)and two (Fig. 3C)dimensional SDS-PAGE, followed by immunoblotting (Fig. 3, B and D). A major immunoreactive band with an approximate M, of 14,000 and a p1 of 8.2 (protein 1) was detected with the antipeptide ALP antibody, but not with control preimmune sheep serum (data not shown). Several lower M, proteins (proteins 2, 3, and 4) which reacted with anti-ALP antibody were also apparent; these may represent proteolytic fragments of ALP. The major immunoreactive protein was subjected to amino acid sequencing. The N-terminal sequence of 18 amino acid residues (Table 1) corresponds exactly to that predicted from the nucleotide sequence of cloned porcine uterine ALP cDNAs (3). Comparison of this sequence with the corresponding sequence for the human protein (10,26) revealed 44% identity within this region. The ability of uterine ALP to inhibit elastase activity was examined following previously described protocols (23). Increasing concentrations of the partially purified protein from AF (Fig. 3) correspondingly decreased available elastase, as measured by residual elastase activity toward its synthetic substrate (Fig. 4). Tissue distribution

and ontogeny of ALP antigen

The data in Table 2 indicate levels of immunoreactive ALP in pig endometrial, myometrial, and placental cytosolic extracts and in pig AF, measured by a RIA using the antipeptide antibody and radiolabeled ALP peptide. Cytosolic extracts of endometrium exhibit low levels of immunoreactive ALP in early pregnancy (day 12), which increased (P = 0.001) to peak levels by midpregnancy (days 60 and 75) and dropped (P = 0.045) to approximately one fifth of the peak concentrations by late pregnancy (days 90 and 105). The placental and myometrial ALP content during pregnancy remained invariant and did not follow the pattern in the corresponding endometrium. AF contained the highest levels of ALP antigen of the samples tested, when expressed on a per mg protein basis. ALP levels in these fluids were highest (P = 0.003) on days 60-90 of pregnancy, but declined (P = 0.001) by day 105, similar to those in the endometrium (Table 2). Cellular localization

of ALP

The localization of ALP in endometrium, placenta, and fetal liver was examined by immunocytochemistry, using the purified IgG fraction of sheep antiserum. The results shown in Fig. 5 are from the endometrium of a

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ANTIPEPTIDE

FIG. 3. Partial purification and immunoblot analysis of uterine ALP on oneand two-dimensional PAGE. Top panel, Gel filtration chromatography of AF from a day 75 pregnant gilt on a Sephadex G-100 column. Column fractions were assayed for the presence of ALP by RIA, as described in the text. Bottom panels: A, One-dimensional pattern of proteins in fractions 36-41 obtained from Sephadex G-100 chromatography of AF from a day 75 pregnant gilt and in crude AF (AF 1 and 2). Proteins were separated in a 15% acrylamide-SDS gel and stained with Coomassie blue. B, Immunoblot analysis of proteins shown in A using the anti-ALP peptide antiserum at a dilution of 1:500. C, Two-dimensional analysis of proteins from Sephadex G-100 fraction 41. Isoelectric focusing was carried out using LKB ampholines in the NEPHGE system, and two-dimensional SDS-PAGE was run in a 20% acrylamide gel. Proteins were stained with Coomassie blue. Proteins 1, 2, 3, and 4 represent protein spots reactive with the antibody in the immunoblots. The relative pH range is designated from 7.1-9.1. The M, of standards (Sigma) are indicated on the left. D, Immunoblot pattern of a gel identical to that in C.

TABLE 1. NH*-Terminal human lung ALP

Pig uterine ALP Human SLPI

ANTIBODIES

0

TO

UTERINE

20

10

30

O&80 -

A. (Kda)

1 38

39

60

70

60

lmmunoreaotlve

ALP(u~/ml)l

no. 40

AF

M r (Kda) 9.1

4111

---_ __f _ ._

7.1

PI

34 24 -

45

/

1s -

34 24 18 14 -

14 -

3

D.

6. Fraction 138

39

no. 40

amino acid sequences of pig uterine and

AENAL KGGAC PPRKI VQC SGKSP KAGVC PPKKS AQC

SLPI, Secretory leukocyte protease inhibitor

60

number

C. Fraction

Mr

1961

40

Fraction +

ALP

(10, 26).

day 60 pregnant pig and the liver of a day 75 fetus, although similar patterns were obtained from endometrium of a day 75 pregnant pig or a day 60 fetus. Immunostaining was localized within the glandular and surface epithelium, but not in the endometrial stroma (Fig. 5, a and b). Specific staining was detected in the placenta (Fig. 5a) and, to a lesser extent, in liver blast

411

AF [

1

2 I

cells (early blood cells; Fig. 5~). Adjacent sections similarly treated with preimmune sheep IgG showed no staining (Fig. 5, corresponding right panels). Maternal

transport of ALP

The presence of high levels of immunoreactive ALP in the placenta, a tissue that does not synthesize ALP (ll), suggests transport of ALP from the endometrium to the feto-placental unit. To determine whether maternal ALP gains access to fetal tissues, levels of this protein were measured in fetal cord and fetal liver (Table 3). Liver cytosolic extracts from day 60, 75, 90, and 105 fetuses had low concentrations of ALP (0.04-0.05 pg/mg protein); these were at least go-fold lower than those in

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ANTIPEPTIDE

1962 100

ANTIBODIES 0

0

a0 l

G; 0 .= m

/

60 0

/

40

z 20 / O#

10

0

20

30

CONCENTRATION

40

4

!50

(rig/ml)

hG. 4. Elastase inhibitor activity of pig uterine ALP. Increasing concentrations of fraction 41 (see Fig. 3) were tested for elastase inhibitor activity, as described in the text. Percent inhibition was calculated relative to the activity of elastase (taken as 100%) in the absence of added fraction 41.

placenta at corresponding days of gestation. Fetal cord blood from mid- to late gestation had detectable levels of ALP which declined in close correlation with levels of uterine ALP (Table 2). Maternal serum levels of ALP were undetectable throughout pregnancy (data not shown). Discussion

The high level of expression of mRNA for the elastase/ cathepsin-G protease inhibitor ALP in pig uterus during mid- and late pregnancy (3) and its apparent limited expression in uteri of animals with an epitheliochorial placenta (27) suggest a stage of pregnancy-dependent role for this protease inhibitor in feto-maternal interactions. In the present study we used a monospecific polyTABLE

2. Ontogeny of immunoreactive Day of pregnancy 12

30 45 60 75 90 105

TO UTERINE

EndoVolMO*No4

ALP

clonal antibody raised against a synthetic peptide corresponding to a hydrophilic region of pig ALP as a tool to elucidate the sites of action of ALP within the microenvironment of the developing fetus. Our results demonstrate that expression of ALP in the uterus is associated with its concomitant transport to the feto-placental unit and presence in the fetal circulation. Similar to the transplacental iron transport protein UF (28), ALP is stored in the allantoic sac; however, the relatively low levels of ALP in the fetal liver suggest that it is not directed primarily to this tissue, which is the major target organ for UF in its capacity as a hematopoietic growth factor (29). We have proposed that ALP may be involved in the establishment and maintenance of the noninvasive epitheliochorial placenta (27). Endometrium from horse (27) and cow (Simmen, R. C. M., and M. J. Fields, unpublished observations), which have epitheliochorial placenta, express high levels of ALP mRNA, in contrast to endometrium from rats, which have hemochorial placenta. The invasive ability of trophoblasts from species with either hemochorial (30, 31) or epitheliochorial (32) placentation suggests that control of physiological processes regulating invasiveness must reside in part within the uterus (1). One potential mechanism could involve timely production by the endometrium of protease inhibitors that can block the inherent ability of trophoblasts to degrade the epithelium, basement membrane, and underlying stroma. In this regard, we have demonstrated endometrial production of ALP during the preimplantation period (day 12 of pregnancy), although the levels were lower than those obtained at midpregnancy. These findings, however, are consistent with additional roles for ALP at mid- and late pregnancy, especially in light of our demonstration of high levels of ALP in fetal, but

ALP in maternal uterus and placenta and in AF during gestation in the pig Tissue or fluid

Endometrium 0.04 f 0.01 (3) (0.037/0.050) ND

Myometrium

Placenta

AF

ND

ND

ND

ND

ND

0.99 f 0.49 (3) (0.30/1.39) 1.52 f 0.57 (3) (1.03/2.32) 4.26 f 2.12 (3) (1.39/6.44) 0.55 + 0.18 (3) (0.31/0.69) 0.59 2 0.20 (3) (0.36/0.80)

0.94 (1)

4.78 f 2.69 (3) (1.97/8.42) 3.09 f 1.76 (3) (1.30/5.50) 6.32 f 4.56 (3) (2.97/12.8) 2.62 f 1.05 (3) (0.91/5.51) 1.96 (1)

7.67 f 3.05 (3) (4.96/10.98) 5.88 + 1.58 (3) (3.89/7.76) 31.29 f 10.26 (3) (22.2114564) 13.33 * 2.10 (3) (10.47/15.48) 18.02 + 7.56 (3) (7.61/25.38) 3.69 f 1.96 (3) (1.55/7&J)

0.35 (1) 0.39 (1) 0.41 (1) 0.28 f 0.01 (2)

Values are expressed as micrograms of ALP per mg total protein (mean rf: SEM; for n = 3, where n is number of gilts). Data in parentheses represent the lowest and highest concentrations of immunoreactive ALP in the samples tested. ND, Not determined.

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ANTIPEPTIDE

ANTIBODIES

TO UTERINE

ALP

1963

FIG. 5. Immunocytochemical staining of pig endometrium and placenta (a), endometrial glands (b), and fetal liver (cl using a purified IgG fraction of the specific antiserum against ALP 16P. Endometrium was obtained from a day 60 pregnant pig, while fetal liver was obtained from a day 75 fetus. Panels on the right correspond to adjacent tissue sections incubated with preimmune sheep IgG. Magnification was x40 for endometrium and fetal liver, and x20 for endometrial glands. Arrows indicate immunoreactive regions within tissue sections.

TABLE 3. Levels of immunoreactive ALP in fetal cord blood and fetal liver during pregnancy in the pig Day of pregnancy 60

Fetal cord blood”

Fetal liver*

2.33 -t 1.45 (3) 0.05 f 0.01 (3) (0.71/4.23) (0.040/0.056) 75 1.99 + 1.40 (3) 0.04 (1) (0.56/3.39) 90 0.15 f 0.03 (3) 0.05 + 0.01 (3) (0.46/0.055) (0.11/0.19) 105 0.17 + 0.06 (3) 0.04 (1) (0.09/0.23) Data in parentheses represent the lowest and highest concentrations of immunoreactive ALP in the samples tested. a Micrograms of ALP per ml (mean + SEM; for n = 3, where n is number of gilts). * Micrograms of ALP per mg total protein (mean f SEM; for n = 3, where n is number of gilts).

not maternal, serum. ALP may placental-uterine interphase at and contribute to the integrity factors that are essential to the

function to maintain the later stages of gestation of proteins and growth growing fetus. Addition-

ally, ALP may be involved in the maturation and function of the fetal lung in its capacity as an inhibitor of proteases produced by leucocytes during increased bronchial activity associated with fetal development (4, 5, 9). Until the present study, the biosynthesis and concomitant localization of ALP protein in any mammalian uterus have not been reported. Results from the present study demonstrate the endometrial and myometrial expression of ALP, consistent with expression of ALP mRNAs in both tissue compartments (11). Interestingly, while levels of endometrial ALP protein appear to change with pregnancy status, myometrial ALP concentrations remained essentially unchanged and lower than those in the corresponding endometrium. This contrasts with the comparable levels of expression for myometrial and endometrial ALP mRNA (11). While it is difficult to examine at the present time the basis for differences in expression owing to the limited numbers of myometrial samples used in the study, our data suggest that myometrial and endometrial expression of ALP protein may be posttranscriptionally regulated. These observations are

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1964

ANTIPEPTIDE

ANTIBODIES

not unique to ALP, since other uterine proteins, such as the type I insulin-like growth factor receptor, appear to be higher in the endometrium than in the corresponding myometrium during early pregnancy in the pig (33), although the levels of expression of the corresponding mRNA in the endometrium and myometrium are similar (34). The subsequent localization of the protein in the placenta, which does not synthesize ALP (ll), suggests that this tissue may be one of the major sites of action of the protease inhibitor during pregnancy. This is consistent with the lack of expression of ALP mRNA in the uterus during the estrous cycle (11). Interestingly, expression of ALP mRNA and protein in the maternal uterus during pregnancy is closely associated with that of another uterine-associated protein, UF (3,16). The spatial and temporal patterns of expression of UF and ALP suggest similar mechanisms of control, which may be pregnancy-dependent. However, UF and ALP mRNAs are differentially controlled by steroid hormones in ovariectomized gilts treated with estrogen and progesterone (11). Thus, pregnancy-associated factors, unrelated to estrogen and progesterone, may be more essential for the controlled expression of these uterine proteins. In this regard, we have demonstrated from transient transfection studies using a steroid-responsive endometrial cell line HRE-H9 (35) that basal UF promoter function is more dependent on nonreceptor tissue-specific factors than on factors involved in steroid induction (36). The amino-terminal sequence of secreted ALP determined from the present study is identical to the protein sequence deduced from the nucleotide sequence of cloned ALP cDNAs (3). The predicted signal sequence for ALP, which is seven amino acids in length, is much shorter than reported concensus signal sequences for eukaryotic proteins (37); however, as shown here, this signal sequence appears functional, allowing the secretion of newly synthesized ALP. In summary, we have shown that ALP secreted by the uterine endometrium during pregnancy is taken up by the placenta and in part by the developing fetus. Although a number of fetal tissues express ALP mRNA in low levels (3), the high level of ALP production by the uterus, which far exceeds that of any maternal or fetal tissues, suggests an important role for ALP in the events surrounding successful pregnancy. The identification of potential specific target sites for the protein provides important insights into the physiological relevance of ALP in species with epitheliochorial placentation. Acknowledgments

The authors thank Dr. Frank A. Simmen for critical review of this manuscript, Dr. Fuller W. Bazer and members of his laboratory for help in the collection of tissues and AF, Cheryl

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Feinstein and Vahideh Lamian for assistance with the art work, and Glenda Walton for secretarial help. We also acknowledge the assistance of the Protein Sequencing and Peptide Synthesis Core facilities of the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR). References 1. Lala PK, Graham CH 1990 Mechanisms of trophoblast invasiveness and their control: the role of proteases and protease inhibitors. Cancer Metastasis Rev 9369-379 2. Morton DB 1977 The occurrence and function of proteolytic enzymes in the reproductive tract of mammals. In: Barrett AJ (ed) Proteases in Mammalian Cells and Tissues. North Holland. New York, pp 445-500 3. Farmer SJ, Fliss AE, Simmen RCM 1990 Complementary DNA cloning and regulation of expression of the messenger RNA encoding a pregnancy-associated porcine uterine protein related to human antileukoproteinase. Mel Endocrinol4:iO95-1104 4. Ohlsson K. Teaner H 1976 Inhibition of elastase from eranulocvtes by the low’moi&ular weight bronchial protease inhib&r. Stand J Clin Lab Invest 36437-445 5. Kramps JA, Franken C, Meyer CJL, Dijkman JH 1981 Localization of a low molecular weight protease inhibitor in serous secretory cells of the respiratory tract. Histochem Cytochem 29:712-716 6. Heinzel R, Appelhans H, Gassen G, Seemuller U, Machleidt W, Fritz H, Steffens G 1986 Molecular cloning and expression of cDNA for human antileukoproteinase from cervix uterus. Eur J Biochem 160:61-67 7. Willems LNA, Kramps JA, Jeffery PK. Dijkman JH 1988 Antileucoprotease in the developing fetal lungs Thorax 43784-786 8. Kramps JA, Franken C, Meijer CJLM, Dijkman JH 1981 Localization of low molecular weight protease inhibitor in serous secretory cells of the respiratory tract. J Histochem Cytochem 29712-719 9. Tegner H, Ohlsson K 1977 Localization of a low molecular weight p&ease inhibitor to tracheal and maxillary sinus mucosa. Hoppe Seylers Z Physiol Chem 358425-429 10. Seemuller U, Arnhold M, Fritz H, Wiedermann K, Machleidt W, Heinzel R, Appelhans H, Gassen HG, Lottspeich F 1986 The acid stable proteinase inhibitor of human mucous secretions (HUSI, antileukoproteinase): complete amino acid sequence as revealed by protein and cDNA sequencing and structural homology to whey proteins and red sea turtle proteinase inhibitor. FEBS Lett 199:4348 11. Simmen RCM, Simmen FA, Bazer FW 1991 Regulation of synthesis of uterine secretory proteins: evidence for differential induction of porcine uteroferrin and antileukoproteinase gene expression. Biol Reprod 44:191-200 12. Hopp TP, Woods KR 1981 Prediction of protein antigenic determinants from amino acid seauences. Proc Nat1 Acad Sci USA 78:3824-3828 13. Kyte J, Doolittle RF 1982 A simple method for displaying the hvdronathic character of a nrotein. J Mol Biol157:105-132 14. Liu F:T, Zinnecker M, Hamaoka T, Katz DH 1979 New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry l&690-697 15. Markwell MAK, Fox CF 1978 Radioiodination of proteins and peptides using iodogen. Pierce Chemical Bull Radioiodination 28 30 16. Simmen RCM, Baumbach GA, Roberts RM 1988 Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein. Mol Endocrinol2:253-262 17. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227680685 18. Roberta RM, Baumbach GA, Buhi AC, Denny JB, Fitzgerald LA, Babelyn SF, Horst MN 1984 Analysis of membrane polypeptides by two-dimensional polyacrylamide gel electrophoresis. In: Venter JC, Harrison L (eds) Molecular and Chemical Characterization of

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cathepsin-G protease inhibitor, antileukoproteinase (ALP): monospecific antibodies to a synthetic peptide recognize native ALP.

Expression of the mRNA encoding the elastase/cathepsin-G protease inhibitor, antileukoproteinase (ALP), is highest in pig uterus during mid- and late ...
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