©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rat Serum Carboxylesterase
CLONING, EXPRESSION, REGULATION, AND EVIDENCE OF SECRETION FROM LIVER (*)

(Received for publication, June 7, 1995)

Bingfang Yan (§) Dongfang Yang Peter Bullock (¶) Andrew Parkinson (**)

From theDepartment of Pharmacology, Toxicology and Therapeutics, Center for Environmental and Occupational Health, University of Kansas Medical Center, Kansas City, Kansas 66160-7417

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Multiple forms of carboxylesterase have been identified in rat liver, and five carboxylesterases (designated hydrolases A, B, C, S, and egasyn) have been cloned. Hydrolases A, B, C and egasyn all have a C-terminal consensus sequence (HXEL) for retaining proteins in the endoplasmic reticulum, and these carboxylesterases are found in rat liver microsomes. In contrast, hydrolase S lacks this C-terminal consensus sequence and is presumed to be secreted. In order to test this hypothesis, a polyclonal antibody was raised against recombinant hydrolase S from cDNA-directed expression in Escherichia coli. In addition to hydrolases A, B, and C (57-59 kDa), this antibody recognized a 67-kDa protein in rat liver microsomes and a 71-kDa protein in rat serum. The 71-kDa protein detected in rat serum was also detected in the extracellular medium from primary cultures of rat hepatocytes. Non-denaturing gel electrophoresis with staining for esterase activity showed that a serum carboxylesterase comigrated with the 71-kDa protein. Immunoprecipitation of the 71-kDa enzyme from rat serum decreased esterase activity toward 1-naphthylacetate and para-nitrophenylacetate. The 71-kDa protein immunoprecipitated from rat serum had an N-terminal amino acid sequence identical to that predicted from the cDNA encoding hydrolase S, providing further evidence that hydrolase S is synthesized in and secreted by the liver. The levels of the 67-kDa protein in rat liver microsomes and the levels of the 71-kDa protein in rat serum were co-regulated. Deglycosylation of microsomes and serum converted the 67- and 71-kDa proteins to a 58-kDa peptide, which matches the molecular mass calculated from the cDNA for hydrolase S. These results suggest that the 67-kDa protein in liver microsomes is a precursor form of hydrolase S that undergoes further glycosylation before being secreted into serum. In rats, liver appears to be the only source of hydrolase S because no mRNA encoding hydrolase S could be detected in several extrahepatic tissues. Serum carboxylesterases have been found to play an important role in lipid metabolism and detoxication of organophosphates, therefore, the secretion of hydrolase S and the modulation of its expression by xenobiotics may have physiological as well as toxicological significance.


INTRODUCTION

Carboxylesterases play an important role in the metabolism of endogenous lipids and foreign compounds, such as drugs and pesticides(1, 2, 3, 4, 5, 6) . In addition to hydrolyzing numerous chemicals, carboxylesterases can catalyze transesterification reactions, which accounts for the conversion of cocaine (a methyl ester) to ethylcocaine (the corresponding ethyl ester) in the presence of an alcohol(7) . Carboxylesterases are abundant cellular components, and they appear to have certain structural roles, which may be important in cellular trafficking. For example, egasyn, a liver microsomal carboxylesterase identified in both rat and mouse, binds to beta-glucuronidase via its active site, which results in sequestration of the protein within the endoplasmic reticulum(8, 9) . In rabbits, microsomal carboxylesterases have recently been shown to interact with and regulate the secretion of acute-phase-response proteins, such as C-reactive protein(10) .

Multiple forms of carboxylesterase have been identified in rat liver microsomes by isoelectrofocusing or non-denaturing gel electrophoresis followed by staining for esterase activity(11, 12) . Previous studies from this laboratory led to the purification and characterization of two carboxylesterases in rat liver microsomes(12, 13) . These two carboxylesterases, designated hydrolases A and B, appear to be the only abundant enzymes in rat liver microsomes that rapidly hydrolyze para-nitrophenylacetate. High levels of hydrolase A are present in testis, whereas high levels of hydrolase B are present in kidney, as determined by enzyme kinetics and Western immunoblotting (12, 13) . We have recently isolated three full-length cDNAs from libraries prepared from rat testis, kidney, and liver. These cDNAs apparently encode hydrolase A, B, and a third microsomal carboxylesterase designated hydrolase C(14, 15, 16) . Our initial attempts to clone hydrolases A and B by immunoscreening a rat liver cDNA library with antibodies against the purified proteins invariably resulted in the isolation of a cDNA (partial sequence) encoding a carboxylesterase that is similar but not identical to those present in rat liver microsomes. Other investigators have isolated the same cDNA when screening rat liver cDNA libraries with antibodies against carboxylesterases purified from rat liver microsomes. Consequently, the cDNA isolated by Takagi et al.(17) with antibody against carboxylesterase E1 does not encode this microsomal carboxylesterase, just as the cDNA isolated by Long et al.(18) does not encode the microsomal carboxylesterase they identified as a target for alkylation by halothane. (^1)These cDNAs encode the same protein, which is hereafter designated hydrolase S.

Carboxylesterases are widely distributed throughout the body and are present in several subcellular organelles, but the highest concentration of carboxylesterases is found in liver microsomes(12) , where they are loosely bound to the luminal surface of the endoplasmic reticulum. The retention of soluble proteins in the lumen of the endoplasmic reticulum of yeast and animal cells is specified by the COOH-terminal consensus sequence, HXEL or KXEL(19, 20) . Hydrolases A, B, and C all have the COOH-terminal HXEL consensus sequence and are found in rat liver microsomes(15) . In contrast, hydrolase S lacks this COOH-terminal consensus sequence and is presumed to be secreted. It is believed that HXEL-containing proteins bind ERD2 (for endoplasmic reticulum retention) or related proteins in the cis-Golgi apparatus, which leads to retrograde transport of HXEL-containing proteins to the endoplasm reticulum(21, 22) . Recent studies have shown that retention or secretion of certain animal proteins is cell typedependent. For example, protein disulfide-isomerase is exclusively localized within the lumen of the endoplasmic reticulum and nuclear envelope in rat hepatocytes, whereas this protein is secreted by rat pancreatic cells(23, 24) . Similarly, cDNA directed expression of thioredoxin and interleukin-1beta in COS-7 cells results in the secretion of thioredoxin but not of interleukin-1beta, although both proteins are secreted by monocytes (25) .

The aim of the present study was to examine the cellular trafficking of hydrolase S and to test the hypothesis that hydrolase S is a serum carboxylesterase synthesized in and secreted by rat liver. We describe the preparation of a high-affinity antibody against recombinant hydrolase S and demonstrate the presence of hydrolase S in rat serum (in vivo) and the secretion of hydrolase S by primary cultures of rat hepatocytes (in vitro). We also present evidence that hydrolase S exists in two forms: a 67-kDa protein, which is the nascent form in liver microsomes, and a 71-kDa protein, which is the mature form in serum. Both proteins are extensively glycosylated. In rats, the liver appears to be the only source of hydrolase S, inasmuch as no mRNA coding for hydrolase S or the 67-kDa protein could be detected in several extrahepatic tissues.


MATERIALS AND METHODS

Chemicals and Supplies

Yeast extract and tryptone were purchased from Difco (Detroit, MI); agarose (medium EEC) was from Midwest Research Institute (St. Louis, MO); collagenase was from Worthington; fetal bovine serum was from JRH Biosciences (Lenexa, KS), and Chee's medium and insulin were from Life Technologies, Inc. Rabbit antibody against rat serum albumin was purchased from Calbiochem; horseradish peroxidase-conjugated, goat anti-rabbit IgG and rabbit peroxidase anti-peroxidase-complex were from Cappel-Organon Teknika Co. (Durham, NC). Sequenase Kit 2.0 was purchased from United States Biochemical (Cleveland, OH), and all restriction enzymes were purchased from Promega Corporation (Madison, WI). Sodium phenobarbital was purchased from Spectrum Chemical Manufacturing. Corp. (Gardena, CA); sodium pentobarbital was from Anthony Products, (Arcadia, CA), and perfluorodecanoic acid was from Aldrich. Pregnenolone 16alpha-carbonitrile was synthesized and purified as described by Sonderfan and Parkinson(26) . Aroclor 1254 was kindly provided by Dr. Stephen Safe (Texas A & M, College Station, TX). Unless otherwise indicated, all other reagents were purchased from Sigma. Sprague-Dawley rats were purchased from Sasco (Omaha, NE).

Cloning the Entire Coding Sequence of Hydrolase S

A cDNA encoding hydrolase S was cloned from a rat liver cDNA library (Clontech, Palo Alto, CA) by two-step PCR (^2)as described previously(16) . The primers were synthesized based on the nucleotide sequence reported by Takagi et al. (17) and Long et al.(18) . The PCR reactions were performed with a Thermocycler 480 (Perkin-Elmer) in a volume of 50 µl containing 25 mM Tris-HCl (pH 8.8 at room temperature), 50 mM KCI, 1.5 mM MgCI(2), 200 µm of each deoxynucleotide, 0.1% Triton X-100, 25 pmol of each primer, and 2 units of Taq polymerase. The first step of PCR amplification was conducted with external paired primers and 0.3 µg of recombinant phage DNA from the liver cDNA library. The second step was conducted with internal paired primers and 0.5 µl of the amplified mixture from the first step. The temperature profile was as follows: 94 °C (1 min), 55 °C (1 min), and 72 °C (2 min) for a total of 25 cycles. The internal paired primers span a 1678-base pair region encoding the entire protein. The sense primer of the internal pair was attached to an EcoRI site, whereas the antisense primer was attached to an XbaI site, which is underlined. The internal primers are as follows: sense, 5`-TCA GAA TTC TGT TCT TCC GCC ATG TGG CTC-3`(88-108); antisense, 5`-GAC TCT AGA AGG CTG GCA GAG ACC CAT TTA-3` (1747-1766). The external primers are as follows: sense, 5`-TTC ATC ACC ATG GTC TCC-3` (53-70); antisense, 5`-TTT GCT CCT CTG GTA CTG-3` (1767-1784).

The PCR-amplified cDNA was digested with EcoRI and XbaI and separated electrophoretically on a 1.2% agarose gel. The cDNA was recovered with a Qiaex agarose gel extraction kit (Qiagen Inc., Chatsworth, CA), inserted into EcoRI-XbaI-digested pUC18 plasmid, and sequenced with a Sequenase 2.0 kit (U. S. Biochemical Corp.) as described previously(14) .

Production of Recombinant Hydrolase S

The production and purification of recombinant hydrolase S was carried out with a protein fusion and purification kit purchased from New England BioLabs, Inc. (Beverly, MA), essentially as described by the manufacturer. The cDNA encoding hydrolase S was removed from pUC18 plasmid and inserted into the pMAL expression vector downstream from the malE gene, which encodes maltose-binding protein, and was expressed in Escherichia coli. The resultant fusion protein (i.e. maltose-binding protein linked to hydrolase S via a factor Xa protease site) was purified by amylose affinity chromatography, and cleaved with factor Xa to release recombinant hydrolase S. Maltose-binding protein was removed on a second affinity column. The recombinant hydrolase S was recovered in the void volume and concentrated by ultrafiltration with a Diaflo PM-30 membrane (Amicon Inc., Beverly, MA).

Production and Purification of Antibodies against Hydrolase S, A, and B

Polyclonal antibodies against recombinant hydrolase S were raised in three male New Zealand White rabbits (Small Stock Industries, Pea Ridge, AK). For the first immunization, each rabbit was injected in the footpads with 150 µg of purified hydrolase S emulsified with an equal volume of Freund's complete adjuvant (Difco). The second immunization was conducted 2 weeks later, at which time the purified protein was emulsified in Freund's incomplete adjuvant. Two weeks after the second immunization, the rabbits received weekly intravenous injections of purified hydrolase S (200 µg/rabbit) via the ear vein. Antibody titer was monitored by Ouchterlony double immunodiffusion, essentially as described by Morgan et al.(13) , and high titer antiserum was collected from the ear artery 7 days after the last boosting. The IgG fraction was purified from high titer antiserum by Protein A column chromatography with Protein A-agarose (Calbiochem) as described by the manufacturer.

Hydrolases A and B were purified from rat liver microsomes, as described by Morgan et al.(12) . Polyclonal antibodies against the purified proteins were raised in rabbits and were subjected to immunoabsorption chromatography to remove cross-reacting antibodies, as described by Morgan et al.(13) . After immunoabsorption, the antibody against hydrolase A was specific for hydrolase A, whereas the antibody against hydrolase B recognized both hydrolase B and C, which are 96% identical in amino acid sequence(16) . Antibody against maltose-binding protein was purchased from New England BioLabs.

Non-denaturing Gel Electrophoresis

Proteins were subjected to non-denaturing gel electrophoresis and stained for esterase activity as described by Morgan et al.(12) with slight modifications. The stacking gel and separating gel contained 3.5 and 7.5% acrylamide, respectively, both at pH 9.0. Electrophoresis was carried out at a constant current of 10 mA for 2 h and then 25 mA for 4 h. After electrophoresis, proteins were stained for esterase activity as described previously(12) , or they were transferred electrophoretically to Immobilon for immunostaining.

Preparation of Liver Microsomes and Sera

In addition to hydrolases A, B, and C, the antibody against recombinant hydrolase S recognized a 67-kDa protein in rat liver microsomes and a 71-kDa protein in rat serum (see ``Results''). It was presumed that the 67-kDa protein was the precursor form of the 71-kDa protein. To test this hypothesis, the co-regulation of these two proteins by xenobiotic treatment was examined. Mature Sprague-Dawley rats (five per group) were treated with known liver microsomal enzyme inducers as described previously(12, 27) . All rats were housed in an American Association for Accreditation of Laboratory Animal Care accredited facility and allowed free access to Purina Rodent Chow 5001 and water. Liver and trunk blood were collected 24 h after the last injection. Blood samples were allowed to clot at 4 °C overnight. Serum was removed and centrifuged at 1,500 g for 10 min to remove cellular debris, pooled, and stored at -80 °C. Liver microsomes were prepared as described by Lu and Levin (28) and stored at -80 °C as suspensions in 250 mM sucrose. The concentration of protein was determined with a commercially available kit (BCA Protein Assay, Pierce) with bovine serum albumin as standard, as described by the manufacturer.

Partial Purification and Immunoprecipitation of Serum Hydrolase S

Hydrolase S was partially purified from rat serum by ammonium sulfate fractionation and anion-exchange chromatography. A saturated solution of ammonium sulfate (at room temperature) was added dropwise to 10 ml of rat serum to give a final concentration of 30% saturation. After stirring slowly for 30 min at 4 °C, the sample was centrifuged at 10,000 g for 30 min at 4 °C. Additional saturated ammonium sulfate was added to the supernatant fraction to give a final concentration of 60% saturation, and the sample was stirred and then centrifuged as above. The pellet was resuspended in 10 ml of phosphate-buffered saline and dialyzed overnight at 4 °C against 1 liter of buffer A (10 mM Tris-HCl, pH 7.4, containing 0.2 mM EDTA). The dialyzed sample was diluted with equal volume of buffer A and applied at 4 °C to a Whatman DE52 anion-exchange column (2.5 20 cm), which had been conditioned previously with a reverse linear gradient of 400 ml of high salt buffer (buffer A containing 1 M NaCI) versus an equal volume of buffer A. After the sample was loaded, the column was washed with buffer A until no more protein was detected in the eluate at 280 nm. The column was eluted with a linear gradient of 200 ml of buffer A versus an equal volume of high salt buffer. Column fractions (1.5 ml) were screened by SDS-PAGE and immunoblotting with antibody against hydrolase S, and those fractions containing partially purified hydrolase S were pooled. The concentration of protein was estimated from the absorbance at 280 nm, based on an extinction coefficient of 1.3 for 1 mg/ml(12) . A final concentration of 20% glycerol was added prior to storing the partially purified preparation of hydrolase S at -80 °C.

Hydrolase S was immunoprecipitated from the partially purified preparation with antibody against the recombinant protein coupled to Protein G-agarose. Protein G-agarose (50 µl) was washed three times with buffer B (10 mM sodium phosphate, pH 7.2, containing 150 mM NaCI) by centrifugation at 500 g for 2 min/wash. After the last wash, the Protein G-agarose pellet was resuspended in 0.5 ml of buffer B containing either no antibody (negative control) or 0.15 mg/ml of antibody against hydrolase S or rat serum albumin (i.e. an irrelevant antibody). After a 4-h incubation at 4 °C with gentle agitation, the antibody-Protein G complexes were washed three times as described above. Following the last wash, 100 µl of partially purified hydrolase S (about 500 µg of protein) and 400 µl of buffer B were added to each tube. After an overnight incubation at 4 °C with gentle agitation, the samples were centrifuged at 3000 g for 5 min at 4 °C. The supernatant fractions were analyzed by Western immunoblotting with antibody against hydrolase S and assayed for enzymatic activity toward para-nitrophenylacetate and 1-naphthylacetate as described by Morgan et al.(12) .

Secretion of Hydrolase S by Primary Cultures of Rat Hepatocytes

Hepatocytes were isolated from mature male Sprague-Dawley rats by the method of Seglen(29) , as modified by Waxman et al.(30) . Hepatocytes (3 10^6 cells) were seeded on rigid (denatured) type 1 collagen (Vitrogen) in 6-cm Lux plastic tissue culture dishes (Nunc, Naperville, IL) in modified Chee's medium containing 100 nM insulin, as described by Waxman et al.(30) . Cells (in the serum-containing media) were allowed to attached for 3 h at 37 °C in 95% relative humidity and 5% CO(2), and were then overlaid with extracellular matrix (matrigel), as described by Sidhu et al.(31) . The medium was then replaced with serum-free modified Chee's medium, which was changed once again on the second day. The cultures were incubated under the same conditions for 3 days. Culture medium was collected, centrifuged at 2,000 g for 10 min to remove debris, and stored at -20 °C until analyzed for the presence of hydrolase S by Western immunoblotting.

Enzymatic Deglycosylation

Deglycosylation was conducted with endoglycosidases H and F (Boehringer Mannhein). Rat liver microsomes or rat serum was denatured in the presence of 100 mM 2-mercaptoethanol and 0.2% SDS at 95 °C for 2 min(32) . For endoglycosidase H, samples from liver microsomes (15 µg) or serum (16 µg) were incubated in 15 µl of 50 mM potassium acetate (pH 5.5) and 1.0 milliunits of enzyme. For endoglycosidase F, serum samples (16 or 4 µg) were incubated in 15 µl of the same buffer plus 20 mM EDTA, 0.6% Nonidet P-40, and 3.0 milliunits of enzymes. After a 20-h incubation at 37 °C, the samples were analyzed by Western immunoblotting.

Other Assays

Proteins were subjected to SDS-PAGE according to the method of Laemmli(33) . Western immunoblotting with antibodies against hydrolase A, B, or S was conducted as described by Morgan et al.(12) . Northern blotting with cDNA encoding hydrolase S was conducted as described previously(14) .


RESULTS

PCR Cloning of Hydrolase S from the Rat Liver cDNA Library

Screening a rat cDNA library by two-step PCR yielded a 1678-base pair cDNA (including primer sequence) encoding the entire polypeptide of hydrolase S. This cDNA was cloned into pUC18, and three clones were sequenced. The sequence of this cDNA was identical to that of the partial cDNA reported by Long et al.(18) , with the exception of 10 nucleotide substitutions. It was also identical to the complete cDNA sequence reported by Takagi et al.(17) , with the exception of six nucleotide substitutions. Four of the seven nucleotide substitutions resulted in alterations in the deduced amino acid sequence. As summarized in Table1, hydrolase S is 74-79% identical to hydrolases A, B, C, and egasyn at the nucleotide sequence level and 67-73% identical at the amino acid sequence level.



Production of Recombinant Hydrolase S

The cDNA encoding hydrolase S was subcloned into a pMAL expression vector downstream from the malE gene, which encodes maltose-binding protein, and was expressed in E. coli. The resultant fusion protein (i.e. maltose-binding protein linked to hydrolase S via a factor Xa protease site) was purified by amylose affinity chromatography and cleaved with factor Xa. As shown in Fig.1, the fusion protein had an apparent molecular mass 100 kDa, based on SDS-PAGE, and was cleaved by factor Xa into two proteins, one of which co-migrated with maltose-binding protein (42 kDa) and one of which had the predicted size of native (unmodified) hydrolase S (58 kDa). Maltose-binding protein was removed on a second amylose affinity column, and purified recombinant hydrolase S was used to prepare a polyclonal antibody in rabbits.


Figure 1: SDS-PAGE analysis of the maltose-binding protein-hydrolase S fusion protein before and after proteolytic cleavage with factor Xa. E. coli were transformed with an expression plasmid harboring a cDNA encoding hydrolase S linked via a factor Xa cleavage site to the gene encoding MBP, as described under ``Materials and Methods.'' The fusion protein was purified by affinity chromatography on an amylose column and cleaved with factor Xa. After cleavage, hydrolase S was separated from MBP by amylose column chromatography. Samples were subjected to SDS-PAGE, and proteins were stained with Coomassie Blue R250. Lane 1 contained 1 µg of purified, intact fusion protein (MBP linked via the factor Xa site to hydrolase S). Lane 2 contained 2 µg of purified fusion protein cleaved with factor Xa. Lane 3 contained 1 µg of purified hydrolase S, which was recovered in the void volume when Factor Xa-cleaved fusion protein was subjected to amylose column chromatography. Lane 4 contained 0.6 µg of purified hydrolase B. Lane 5 contained 0.4 µg of purified MBP from New England BioLabs. The apparent molecular masses of the intact fusion protein, hydrolase S, and MBP were 100, 58, and 42 kDa, respectively.



Secretion of the 71-kDa Protein (Hydrolase S) by Rat Liver

The secretion of hydrolase S into serum (in vivo) and the medium of cultured hepatocytes (in vitro) was examined by Western immunblotting. As shown in Fig.2(right panel), the antibody against recombinant hydrolase S cross-reacted with purified hydrolases A and B. In addition to hydrolases A and B (57 and 59 kDa, respectively), this antibody recognized a third protein (67 kDa) in rat liver microsomes and recognized an even larger protein (71 kDa) in rat serum. The immunostaining of the 67-kDa protein in liver microsomes was much weaker than that of either hydrolase A or B, indicating that the levels of this protein in the microsomes are relatively low. The 67- and 71-kDa proteins were not recognized by antibodies against hydrolase A or B (Fig.2, left and middle panels), implying that neither hydrolase A nor hydrolase B is secreted into serum. No immunostaining occurred when the immunoblots shown in Fig.2were probed with antibody against maltose-binding protein (data not shown).


Figure 2: Immunoblots of purified hydrolases A and B, rat liver microsomes and rat serum probed with antibodies against hydrolase A, hydrolase B, or recombinant hydrolase S. A mixture of purified hydrolases A and B (0.3 µg each), liver microsomes (20 µg, lane 2), and serum (0.5 µl) was analyzed by Western immunoblotting, as described under ``Materials and Methods.'' The liver microsomes and serum were pooled samples from five Sprague-Dawley male rats (12-week old). Left panel, immunoblot probed with antibody against hydrolase A that was immunoabsorbed against hydrolase B. Middle panel, immunoblot probed with antibody against hydrolase B that was immunoabsorbed against hydrolase A. Right panel, immunoblot probed with a polyclonal antibody against purified, recombinant hydrolase S that cross-reacts with hydrolases A (57 kDa) and B (59 kDa).



As shown in Fig.3, primary cultures of rat hepatocytes secreted a protein recognized by antibody against hydrolase S, and this protein comigrated on SDS-PAGE with the 71-kDa protein in rat serum. This 71-kDa protein was not present in the culture medium itself and was detected in the medium only when hepatocytes were cultured under conditions that restored near normal cell morphology and the expression of liver-specific genes (results not shown). Inasmuch as the hepatocytes were cultured in serum-free medium, the results in Fig.3establish that, if the 67-kDa protein is a precursor to the 71-kDa protein, this conversion occurs in the liver prior to secretion of the 71-kDa protein. In other words, the 67-kDa protein is not secreted and subsequently converted to the 71-kDa protein in the serum.


Figure 3: Secretion of hydrolase S from primary cultures of rat hepatocytes as determined by Western immunoblotting. Hepatocytes from 7- to 8-week old Sprague-Dawley male rats were isolated and cultured in Chee's medium, as described under ``Materials and Methods.'' The secretion of hydrolase S into the culture medium was analyzed by Western immunoblotting with a polyclonal antibody against purified, recombinant hydrolase S that cross-reacts with hydrolases A and B. Lane 1 contained 20 µg of rat liver microsomes (hydrolase A, hydrolase B, and a 67-kDa protein were detected). Lane 2 contained 30 µl of extracellular medium from primary cultures of rat hepatocytes (a 71-kDa protein was detected). Lane 3 contained 0.5 µl of rat serum (a 71-kDa protein was detected). Lane 4, contained 30 µl of Chee's medium prior to culturing rat hepatocytes. Lane 5 contained 0.3 µg of rat serum albumin.



Esterase Activity of the 71-kDa Protein in Serum

To determine whether the 71-kDa protein has esterase activity, rat serum were subjected to non-denaturing gel electrophoresis, followed by staining for esterase activity or immunoblotting with antibody against hydrolase S. As shown in the right panel of Fig.4, the 71-kDa form of hydrolase S in rat serum was electrophoretically distinct from those present in rat liver microsomes as determined by immunoblotting. The 71-kDa protein catalyzed the hydrolysis of 1-naphthylacetate as shown in the left panel of Fig.4.


Figure 4: Immunostaining and esterase activity staining of rat liver microsomes, rat serum, and purified hydrolase A and B after non-denaturing gel electrophoresis. Purified hydrolase A (0.3 µg), purified hydrolase B (0.3 µg), pooled liver microsomes (10 µg), and pooled rat serum (0.5 µl) were subjected to non-denaturing gel electrophoresis as described under ``Materials and Methods.'' Staining for esterase activity was based on the formation of a black, insoluble complex between diazotized Fast Blue RR and 1-naphthol, which was released enzymatically from 1-naphthylacetate (left panel). The identical samples were also subjected to non-denaturing gel electrophoresis and transferred electrophoretically to Immobilon for immunostaining with anti-hydrolase S (right panel).



To determine whether the 71-kDa protein contributes significantly to the esterase activity of rat serum, esterase activity toward 1-naphthylacetate and para-nitrophenylacetate was measured before and after immunoprecipitation of the 71-kDa protein with antibody against hydrolase S. Immunoprecipitation of the 71-kDa protein directly from rat serum caused only a small decrease in the rate of hydrolysis of 1-naphthylacetate or para-nitrophenylacetate, which suggests that enzymes other than the 71-kDa protein in rat serum contribute more to the hydrolysis of these carboxylic esters (results not shown). The 71-kDa protein was partially purified from rat serum by a combination of ammonium sulfate fractionation and anion-exchange chromatography. The final preparation contained at least five proteins in addition to the 71-kDa protein, as determined by SDS-PAGE (results not shown). Immunoprecipitation of the 71-kDa protein from partially purified hydrolase S with antibody against hydrolase S decreased esterase activity toward 1-naphthylacetate and para-nitrophenylacetate by 52 and 22%, respectively, as shown in Fig.5.


Figure 5: Effects of immunoprecipitation of hydrolase S on the hydrolysis of para-nitrophenylacetate and 1-naphthylacetate by partially purified hydrolase S from rat sera. Hydrolase S partially purified from rat sera was prepared by ammonium sulfate and ion-exchange chromatography. The partially purified hydrolase S was incubated overnight at 4 °C with the complex of Protein G-agarose and antibody against hydrolase S, the complex of Protein G-agarose and antibody against rat serum albumin, or the same amount of Protein G-agarose alone as described under ``Materials and Methods.'' After the immunoprecipitates were removed by centrifugation, the supernatant fractions were assayed for hydrolytic activities with 1 mMpara-nitrophenylacetate or 1 mM 1-naphthylacetate as described previously(1) . Rates are expressed relative to control (partially purified hydrolase S + Protein G agarose alone). The supernatant fractions were also analyzed by immunoblotting as described under ``Materials and Methods.''



The 71-kDa protein was isolated from the immunoprecipitate by SDS-PAGE, blotted onto a polyvinyldifluoride membrane, and subjected to protein microsequencing. Nine of the first 10 amino acid residues at the N terminus were unambiguously identified, and the sequence matched that deduced from the cDNA encoding hydrolase S beginning with residue 19, indicating that the signal peptide is cleaved from hydrolase S prior to secretion of the 71-kDa protein.

Coregulation of the 67- and 71-kDa Proteins by Xenobiotic Treatment

The results shown in Fig.2-4 were interpreted as evidence that hydrolase S is synthesized in the liver and undergoes extensive post-translational modification prior to its secretion as a 71-kDa protein in serum. Incomplete processing of hydrolase S was hypothesized to produce the 67-kDa protein in liver microsomes, which is, therefore, a precursor to the 71-kDa serum protein. In support of this hypothesis, the levels of mRNA encoding hydrolase S, the levels of the 67-kDa protein in liver microsomes, and the levels of the 71-kDa protein in serum were coregulated by xenobiotics, some of which induced and some of which suppressed the expression of hydrolase S (Fig.6). The levels of the 67- and 71-kDa proteins were both suppressed by beta-naphthoflavone (BNF, 50-60%), dexamethasone (DEX, >80%), perfluorodecanoic acid (PFDA, 55-60%), and isoniazid (55-60%), but they were both induced by Aroclor 1254, 3-methylcholanthrene (3-MC), pregnenolone-16alpha-carbonitrile (PCN), phenobarbital (PB,) and troleandomycin (TAO) by an approximate 1.7-fold.


Figure 6: Effects of treating Sprague-Dawley rats with various xenobiotics on the expression of microsomal and serum hydrolase S and its mRNA. Male rats (7 weeks old, five per group) and female rats (troleandomycin treatment only) were treated with various xenobiotics at dosages known to induce liver microsomal enzyme inducers, as described under ``Materials and Methods.'' Liver microsomes (25 µg of protein) and serum samples (0.5 µl) were subjected to SDS-PAGE and transferred electrophoretically to Immobilon for immunostaining with a polyclonal antibody against hydrolase S. For Northern blotting analysis, total RNA was isolated from the treated or control rats with a modified acid phenol-chloroform extraction method (14) . Pooled total RNA samples (10 µg) of each group were subjected to 2.2 M formaldehyde-agarose electrophoresis and transferred to Nytran nylon membrane by a vacuum blotting system. The blots were probed with a cDNA encoding hydrolase S, which was radiolabeled with [P]dCTP by random primer extension. To normalize the abundance of 28 S rRNA contained in each sample, the same membrane was stripped by boiling 2 15 min and reprobed with an oligonucleotide (hybridize with 28 S rRNA) radiolabeled with [P]ATP by T4 polynucleotide kinase. Note: the cross-reaction of anti-hydrolase S with hydrolases A and B in rat liver microsomes is not shown to facilitate the comparison between microsomal and serum hydrolase S.



It should be noted that the antibody against hydrolase S also recognized hydrolases A and B (57 and 59 kDa) in rat liver microsomes. The effect of xenobiotic treatment on the expression of these carboxylesterases was previously reported by Morgan et al.(13) , hence, this portion of the immunoblot was deleted from Fig.6for clarity.

Enzymatic Deglycosylation

Treatment of rat liver microsomes with endoglycosidase H decreased the molecular mass of hydrolase A, B, and the 67-kDa protein by 1, 2, and 9 kDa, respectively (Fig.7). After deglycosylation, hydrolase B and the 67-kDa protein apparently comigrated, which is consistent with the fact that the predicted molecular masses for both proteins from cDNAs are 58 kDa(15, 17) . Treatment of serum with endoglycosidase H failed to change the electrophoretic mobility of the 71-kDa protein. However, treatment of serum with endoglycosidase F converted the 71-kDa protein to a 58-kDa protein (Fig.7). The Golgi apparatus is known to modify the carbohydrate chains of secretory proteins by adding or changing sugar moieties(34) . These modifications on the 67-kDa protein presumably results in the serum protein with a higher molecular mass (71 kDa) and form biantennary complex type oligosaccharide, a type of carbohydrate chain which can be cleaved by endoglycosidase F but not by endoglycosidase H(35, 36) .


Figure 7: Changes in the electrophoretic mobility of the 67-kDa protein in microsomes and the 71-kDa protein in serum following deglycosylation with endoglycosidases. Microsomes (25 µg) or serum (16 or 4 µg) pooled from five rats were incubated with 1.0 milliunits of endoglycosidase H (Endo H) or 3.0 milliunits of endoglycosidase F (Endo F) under SDS-denaturing condition at 37 °C for 20 h, as described under ``Materials and Methods.'' The samples were subjected to SDS-PAGE, transferred electrophoretically to Immobilon, and probed with antibody against hydrolase S. The three bands with control microsomal sample are the 67 kDa form of hydrolase S, hydrolase A (57 kDa), and hydrolase B (59 kDa).



Tissue Distribution of Hydrolase S

To determine whether hydrolase S is expressed in extrahepatic tissues, microsomes from kidney, testis, prostate, intestine, lung, heart, and brain were analyzed by immunoblotting with antibody against hydrolase S. None of the extrahepatic tissues examined expressed the 67-kDa protein, as shown in Fig.8. However, as expected, the antibody recognized a 57-kDa protein in those tissues that express hydrolase A, namely liver, testis, lung, and prostate, and recognized a 59-kDa protein in those tissues that express hydrolase B, namely liver and kidney. RNA samples from the same tissues were analyzed by Northern blotting with the cDNA encoding hydrolase S, as shown in Fig.8. Liver was the only tissue examined that expressed the mRNA encoding hydrolase S, just as it was the only tissue that expressed the 67-kDa protein.


Figure 8: Tissue distribution of microsomal hydrolase S as determined by Western immunoblotting and Northern blotting. Microsomes from various organs were prepared from 7-week-old Sprague-Dawley rats. The microsomal samples (25 µg of protein) pooled from four rats were subjected to SDS-PAGE and transferred electrophoretically to Immobilon for immunostaining. The blots were probed with a polyclonal antibody against hydrolase S that recognizes hydrolase A (57 kDa), hydrolase B (59 kDa), and microsomal hydrolase S (67 kDa) as shown in the top panel. For Northern blot analysis, total RNA was isolated from the treated or control rats with a modified acid phenol-chloroform RAN extraction method(14) . Pooled total RNA samples (10 µg) from five rats were subjected to 2.2 M formaldehyde-agarose gel electrophoresis and transferred to Nytran nylon membrane by a vacuum blotting system. The blot was probed with a cDNA encoding hydrolase S, which was radiolabeled with [P]dCTP by random primer extension as described under ``Materials and Methods.''




DISCUSSION

The tetrapeptide sequence KXEL or HXEL is found at the carboxyl terminus of luminal and some transmembrane proteins in the endoplasmic reticulum. This sequence is necessary to ensure the retention of proteins in the lumen of the endoplasmic reticulum despite the high rate of forward vesicular transport to the Golgi apparatus and subsequent organelles of the secretory pathway(19, 20, 39, 40) . Hydrolases A, B, and C all have this COOH-terminal HXEL consensus sequence and are found in rat liver microsomes(15) . In contrast, hydrolase S lacks this COOH-terminal consensus sequence and has been postulated to be secreted into blood. The results of the present study support this hypothesis inasmuch as Western immunoblotting with antibody against recombinant hydrolase S detected a 71-kDa protein in both serum and the medium of cultured hepatocytes. The 71-kDa protein in serum has esterase activity toward 1-naphthylacetate and para-nitrophenylacetate ( Fig.4and Fig. 6), consistent with this protein being a carboxylesterase. Furthermore, the NH(2)-terminal amino acid sequence of the 71-kDa protein is identical to that deduced from the cDNA encoding hydrolase S.

The 67-kDa protein in liver microsomes recognized by the antibody against recombinant hydrolase S is likely the precursor form of hydrolase S (71 kDa) inasmuch as these two proteins were coregulated in rats treated with 11 different xenobiotics (Fig.6). The 67-kDa protein is likely the newly synthesized protein of hydrolase S in the rough endoplasmic reticulum, rather than a protein that is transported back from the cis-Golgi apparatus like HXEL-containing proteins. Several lines of evidence from the present study support this interpretation. Based on Western immunoblotting, the level of the 67-kDa protein was only 5% of the levels of hydrolases A and B. Such a low level likely corresponds to the newly synthesized protein. Furthermore, changes in the level of the 67-kDa protein paralleled changes of the level of mRNA for hydrolase S, suggesting that the accumulation of the 67-kDa protein in microsomes depends on the rate of synthesis of hydrolase S (Fig.6). Deglycosylation studies indicated that the hydrolase S peptide (58 kDa) is converted to a 67-kDa protein by N-linked glycosylation (Fig.7), which takes place in the rough endoplasmic reticulum as seen with other carboxylesterases(41) . During its passage through the Golgi apparatus and secretory pathway, hydrolase S is apparently glycosylated further to produce the 71-kDa protein secreted into serum.

Glycosylation of hydrolase S increases its apparent molecular mass by 20% (from 58 to 71 kDa), whereas glycosylation increases the size of rat microsomal carboxylesterases (e.g. hydrolases A, B, and C) by less than 5%(17) . The higher degree of glycosylation of hydrolase S is consistent with the fact that hydrolase S contains five putative N-linked glycosylation sites, whereas the rat liver microsomal carboxylesterases contain no more than three(14, 15, 16) . Other secretory proteins such as coagulation factors V and VIII have been shown to contain both N- and O-linked carbohydrate chains(42) . However, hydrolase S apparently contains only N-linked carbohydrate chains because treatment of serum with endoglycosidase F converted the 71-kDa protein to a 58-kDa peptide. Slight differences in the degree to which hydrolase S is glycosylated may be responsible for microheterogeneity of serum hydrolase S, which appeared as a diffusive band on Western immunoblots ( Fig.2and Fig. 5). Glycosylation of hydrolase S may be required for the secretion of this protein, as previously shown for coagulation factors V and VIII(42) . Extensive glycosylation of rat hydrolase S may be also required for its enzymatic activity and stability in serum, inasmuch as the recombinant hydrolase S produced in E. coli was devoid of esterase activity (both before and after cleavage of the fusion protein), and cleavage of the fusion protein with factor Xa decreased the stability of hydrolase S (data not shown).

We have recently attempted to express the cDNA encoding hydrolase S in a baculovirus/Sf21 system to study the catalytic properties of hydrolase S. The Sf21 cells infected with recombinant baculovirus, but not the cells infected with wild-type baculovirus, synthesized a truncated form of hydrolase S. Unexpectedly, this truncated form of hydrolase S was retained in the endoplasmic reticulum. Other cell lines have also failed to correctly process hydrolase S. COS cells transfected with plasmid containing a cDNA encoding hydrolase S produced a carboxylesterase that did not comigrate with any of the carboxylesterases present in rat serum, as determined by non-denaturing gel electrophoresis with staining for esterase activity (43) or by Western immunoblotting(44) . It is not unprecedented for cells to differ in their ability to process secretory proteins. For example, endoplasmin (a protein related to glucose-regulated protein) is present in the microsomal fraction of rat hepatocytes, whereas it is secreted by rat pancreatic cells(24) . These findings suggest that primary cultures of rat hepatocytes may be required to establish the trafficking mechanisms that govern whether hydrolase S is retained or secreted by the liver. In this regard, it is significant that we established cell culture conditions that support the secretion of hydrolase S from rat hepatocytes.

The antibody against hydrolase S cross-reacted with hydrolases A and B, which was anticipated based on the structural relatedness of these carboxylesterases (Table1). Although antibodies against hydrolases A and B did not recognize the 67-kDa protein in microsomes or the 71-kDa in serum (Fig.2), they recognized recombinant hydrolase S produced by E. coli (data not shown). We and others have attempted to clone hydrolases A and B from rat liver cDNA libraries with antibodies against these microsomal proteins, but succeeded instead in cloning hydrolase S(17, 18, 45) . The results of the present study provide a likely explanation for this phenomenon. The mRNA encoding hydrolase S is abundant and probably well represented in cDNA libraries prepared from rat liver. When synthesized by E. coli, hydrolase S is not glycosylated and can be recognized by the antibodies against microsomal carboxylesterases. This interpretation is reinforced by our successful cloning of hydrolases A and B from cDNA libraries prepared from rat testis and kidney(14, 15) , respectively, two tissues that contain high levels of microsomal carboxylesterases but do not express hydrolase S.

The susceptibility of animals to organophosphate toxicity is inversely related to serum carboxylesterase levels(46) . Previous studies have shown that phenobarbital treatment protects rats from parathion toxicity(47) , whereas treatment with beta-naphthoflavone has the opposite effect(48) . The results of the present study show that treatment of rats with phenobarbital increases the serum level of hydrolase S, whereas treatment with beta-naphthoflavone decreases it. This suggests that hydrolase S may play a role in detoxifying organophosphates although further studies are required to test this possibility. Hydrolase S may also have physiological functions. In rats, fasting followed by refeeding with a fat-free diet significantly decreased serum esterase activity(35) . In mice, a serum carboxylesterase (pEs-N) has been shown to rapidly hydrolyze steroid hormone esters(49) . Therefore, hydrolase S may play an important role in lipid and steroid hormone metabolism. The mechanisms by which xenobiotics regulate the levels of serum carboxylesterase are not known. The results of the present study show that xenobiotic-induced changes of the 67 and 71 kDa forms of hydrolase S reflect increase or decrease in mRNA levels. This suggests that hydrolase S is regulated by xenobiotics at the levels of transcription and/or mRNA turnover.


FOOTNOTES

*
This work was supported by Grant ES 04996 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a grant from the Procter & Gamble Company as part of its International Program for Animal Alternatives.

Supported by a grant from the Procter & Gamble Company as part of its International Program for Animal Alternatives. Present address: Ocean Chemistry Division, Institute of Ocean Sciences, P. O. Box 6000, 9860 W. Sannich Road, Sidney, British Columbia V8L5T6, Canada.

**
To whom correspondence should be addressed: Dept. of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160-7417. Tel.: 913-588-7512; Fax: 913-588-7330.

^1
Based on molecular weight, isoelectric point, NH(2)-terminal amino acid sequence and substrate specificity, carboxylesterase E1 appears to be identical to hydrolase A, which also appears to be the 59-kDa protein that becomes trifluoroacetylated by halothane (12, 13).

^2
The abbreviations used are: PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


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