Human Egasyn Binds β-Glucuronidase but neither the Esterase Active Site of Egasyn nor the C Terminus of β-Glucuronidase Is Involved in Their Interaction

Archives of Biochemistry and Biophysics Vol. 372, No. 1, December 1, pp. 53– 61, 1999 Article ID abbi.1999.1449, available online at http://www.idealibrary.com on

Human Egasyn Binds b-Glucuronidase but neither the Esterase Active Site of Egasyn nor the C Terminus of b-Glucuronidase Is Involved in Their Interaction M. Rafiq Islam, 1 Abdul Waheed, Gul N. Shah, Shunji Tomatsu, and William S. Sly 2 E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Health Sciences Center, 1402 S. Grand Boulevard, St. Louis, Missouri 63104

Received March 16, 1999, and in revised form August 17, 1999

Lysosomal b-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine b-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human b-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using fulllength mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg–GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that

1 Present address: Department of Chemistry, Northwest Missouri State University, Maryville, MO 64468. 2 To whom correspondence should be addressed. Fax: 314-7761183. E-mail: [email protected].

0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

(i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg. © 1999 Academic Press Key Words: human egasyn; human liver carboxylesterase.

Egasyn (Eg) 3 is an ER-resident protein identified in liver and/or kidney in mouse, rat, rabbit, and frog. It is a bifunctional protein in mouse (1–3). It functions as a nonspecific carboxyl esterase called esterase-22 (5) that likely metabolizes a variety of compounds including herbicides, insecticides, anesthetics, analgesics, monoglycerides, and CoA esters (6). In mouse liver, kidney, and lung, but not in spleen, brain, heart, erythrocytes, testis, and skin, mEg has a second function, namely to bind GUSB and sequester 10 –25% of total mGUSB in the ER. The rest is delivered to lysosomes (1, 7). Such dual localization is unique to GUSB among lysosomal enzymes. In all animals characterized so far, GUSB is a product of a single gene, and the polypeptide contains no classical signal for ER retention (8 –11). The requirement for lysosomal GUSB in the catabolism of a wide variety of glycosaminoglycans is evident 3 Abbreviations used: Eg, egasyn; hEg, human egasyn; mEg, mouse egasyn; GUSB, b-glucuronidase; hGUSB, human b-glucuronidase; mGUSB, mouse b-glucuronidase; rGUSB, rat b-glucuronidase; BPNP, bis-p-nitrophenyl phosphate; ER, endoplasmic reticulum; CRP, C-reactive protein; hCE, human carboxylesterase; MTN, multi-tissue Northern blot; PBS, phosphate-buffered saline.

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from human patients with GUSB deficiency (mucopolysaccharidosis type VII or Sly syndrome) and from the MPS VII mouse (12, 13). However, the function of ER GUSB has remained unclear. A few lines of evidence suggested a possible role in the hydrolysis of endogenous and xenobiotic glucuronides and the regulation of the level of these substrates (14 –17). In mouse, it was demonstrated that an octamer sequence (FGSRPFTF) at the C terminus of the GUSB contains information necessary and sufficient for complex formation with mEg (18). When appended to rat AGP, a secretory protein, this octamer sequence allowed AGP to form a complex with mEg within the ER. The absence of this octamer in the mature form of GUSB (from which the C-terminal 18 –19 residues have been removed enzymatically (19)) explains why the mature lysosomal form of mGUSB does not bind mEg. Although Eg has been shown to exist in several animal species besides rodents, there is no conclusive evidence of its presence in humans. In expression studies, mEg was found not to form a complex with monkey GUSB (20). Moreover, when the C-terminal 7-residue peptide (LENSPFT) from hGUSB was substituted for the mouse octamer, no complex formation between the resultant chimeric GUSB and mEg was observed (20). In this paper, we provide evidence for the existence of a functional homologue of mouse egasyn in humans that has carboxyl esterase activity. We show that the protein encoded by this homologous cDNA specifically interacts with hGUSB when coexpressed in COS cells. However, unlike the requirements for an mEg– mGUSB complex formation, neither the esterase active site of the hEg nor the C-terminal portion of the GUSB appears to be involved in the complex formation with hEg. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg. MATERIALS AND METHODS Cloning, sequencing, and tissue distribution. A human liver cDNA library in the Uni-ZAP XR vector (Stratagene) was screened using mouse egasyn cDNA as a probe (21). A partial cDNA clone was isolated and the missing 59 end was obtained by PCR of the library. First, T3 primer in the vector and a reverse primer (59-CTCCTCCCGCTGACTCTCC-39) 671 bp downstream of the cloned fragment were used for amplification. A band corresponding to the expected size of ;700 bp was cut from the gel, extracted, and used as a template for the second PCR using T3 and a different reverse primer (59-CCTGCACTTTGCCATGCACG-39) 101 bp downstream from the cloned fragment. Both PCRs were performed in 13 buffer, 2 mM MgCl 2, and 0.2 mM dNTP for 35 cycles of 1 min at 94°C, 2 min at 55°C, and 2 min at 72°C. The amplified fragment was subcloned into pBluescript and sequenced. The unique SacII site (60 bp downstream) in this fragment was utilized to ligate the PCR fragment to the cloned fragment in order to obtain a full-length 1941-bp cDNA. Both the PCR and cloned fragments were sequenced by the dideoxy chain-termination method using 35S-labeled dATP. A multitissue Northern (MTN) blot obtained from Clontech was

probed with hEg cDNA random primer labeled according to the instructions provided by the supplier. Each lane contained 2 mg poly(A) RNA. The MTN blot was also reprobed with b-actin cDNA. Expression in COS-7 cells. The cDNA insert was blunt-ended and subcloned into three different vectors: pCD (22), pJC119 (23), and pCAGGS (24). In all cases, COS cells (0.5 3 10 6 cells) were transfected using the DEAE– dextran procedure as described (18). For native gels and carboxylesterase activity assay, transfected cells were harvested 72 h after transfection in 20 mM imidazole–HCl, pH 7.4, by sonication and then centrifuged at maximum speed in a microcentrifuge for 30 min at 4°C. For b-glucuronidase activity, cells were harvested in 0.3% deoxycholic acid and media collected 76 h after transfection. Enzyme assays. Cell extracts and media were assayed for GUSB fluorometrically using 4-methylumbelliferyl-b-D-glucuronide (Sigma) as substrate (19). Carboxylesterase activity in cell extracts was measured using b-naphthyl acetate and Fast Garnet GBC salt (both from Sigma) as follows: 50 ml cell lysates in 780 ml of b-naphthyl acetate (0.32 mM) and 200 ml of Na 2HPO 4 (0.2 M, pH 7.0) were incubated at 39°C for 10 min. Then 200 ml of 10% SDS containing 1 mg of Fast Garnet GBC salt was added and incubated at room temperature for 10 min. The absorbance of the final mixture was measured at 560 nm (25). Native and SDS– gel electrophoresis. Cell extracts were brought to 0.25 M sucrose and separated on nondenaturing 6% polyacrylamide gels and stained for esterase activity with a-naphthyl acetate/ Fast Blue BB (18). Immunoprecipitates were analyzed by 7.5% sodium dodecyl sulfate–polyacrylamide gels followed by treatment with ENHANCE and fluorography. Cross-linking, immunoprecipitation, and immunofluorescence. For coimmunoprecipitation experiments, COS cells were labeled for 2 h with Tran 35S-Label 60 h posttransfection and harvested by scraping in phosphate-buffered saline (PBS). PBS was removed by centrifugation and the cell pellets were stored at 270°C. For immunoprecipitation, 0.5 ml of 50 mM imidazole buffer, pH 7.4, containing 0.15 M NaCl and 0.5% Triton X-100 was added to the frozen cell pellets and sonicated twice for 10 s. Following centrifugation for 30 min at maximum speed in a microcentrifuge at 4°C, the supernatants were collected. For chemical cross-linking, frozen cell pellets were sonicated in the same solution without Triton X-100 and incubated with 1 mM of thio-cleavable cross-linker dithiobis (succinimidyl propionate) (Pierce) for 30 min at room temperature. Then 0.5 ml of 50 mM Tris–HCl, pH 7.4, containing 0.1% SDS, 0.5% DOC, 1% NP-40, 1 mM MgCl 2, and 20 mM monoethylamine was added and the mixture incubated for 15 min at room temperature. The cells were then sonicated twice for 10 s and centrifuged at maximum speed in a microcentrifuge at 4°C for 30 min. Following preclearing with prewashed Staph A, the supernatant was collected by centrifugation at 15,000 rpm for 1 h and immunoprecipitated using anti-hGUSB antibody, anti-rGUSB antibody, or a mixture of both, depending on the experiments, as described (19). Cells grown on coverslips were fixed in 3% paraformaldehyde, permeabilized with 0.3% Triton X-100 in PBS for 5 min, washed with PBS, and incubated for a total of 15 min in blocking solution containing 0.2% gelatin in PBS. Anti-hCE antibody (4) was diluted in the same blocking solution and incubated with the cells for 2 h. The cells were washed with 0.2% gelatin in PBS and treated with fluorescein-conjugated goat anti-rabbit immunoglobulin (Sigma) for 1 h. BPNP treatment and deglycosylation. Immunoprecipitates resuspended in 60 ml 50 mM Tris–HCl, pH 7.4, were treated with 5 or 50 mM BPNP overnight in the dark at 4 or 37°C. Cross-linked immunoprecipitates were treated with 5 mM BPNP at 4°C in the same solution containing 5% 2-mercaptoethanol. Following treatment with BPNP, the supernatants were separated from the pellets by centrifugation. Both the supernatants and pellets were boiled with

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INTERACTION OF HUMAN EGASYN WITH GLUCURONIDASES 23 SDS sample buffer and briefly centrifuged, and the resultant supernatants were loaded onto gels. For protein N-glycosidase treatment, immunoprecipitates were heated for 5 min in 0.1 M phosphate buffer, 0.1% SDS, 0.6% NP-40, 1% 2-mercaptoethanol, pH 8.6. After centrifugation, the clear supernatant was made with 1 mM phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, 1 mM EDTA and digested with 50 mU of the enzyme for 22 h at 37°C.

RESULTS

Isolation of a full-length cDNA for hEg and comparison of its nucleotide and amino acid sequences. Seven positive clones were identified in 0.5 3 10 6 plaques, of which the longest was found to be nearly full-length (lacking 59-UTR and first two bases of the initiation codon ATG). The 59 end segment was obtained by nested PCR of the same cDNA library using two primers in the coding region and T3 primer in the vector. The 1941-bp full-length hEg cDNA includes a 27-bp 59-UTR, an open reading frame encoding a protein of 566 amino acids with one potential glycosylation site, and a 216-bp 39-UTR containing a polyadenylating signal. The cDNA is almost identical to two previously reported carboxylesterase cDNAs, hCE (GenBank Accession No. L07764) and hCEv (for hCE variant, GenBank Accession No. L07765) from a human liver library (4). For this reason, anti-hCE has been used for immunoprecipitation experiments. The hCEv differs from hCE only in having two 3-bp deletions at positions 52–54 and 1086 –1089 (counting from the initiation ATG of hCE) which result in deletions of Ala-18 and Gln-363 in the hCEv precursor. The hEg cDNA clone we isolated has only the first 3-bp deletion (position 52–54). In addition, it differs from hCE in three ways: (i) deletion of a C at position 24 (4th position upstream of the initiation ATG); (ii) substitution of a G for T at position 5, changing the Trp at position 2 to Leu; and (iii) two substitutions of Gs for Cs at positions 170 and 1610, converting both Gly-57 and Gly-537 to corresponding Ala residues. The hEg has 78% identity in nucleotide sequence with mEg and 78% identity in predicted amino acid sequence. Mouse Eg contains 562 amino acid residues and three potential glycosylation sites. If the signal peptide cleavage site is conserved between mouse and human, cleavage would remove 18 amino acids from the N terminus. The predicted protein sequence of hEg also shows an ER retention signal (HIEL) in the C terminus (HTEL in the mEg). Both sequences are effective signals for ER retention (26 – 28) and we can assume that hEg is retained in the ER by binding to the KDEL receptor. Expression of hEg in COS cells and tissues. The entire hEg cDNA was inserted into expression vector pCD, in which expression is driven by the SV40 late promoter. Mouse Eg cDNA in this vector was used as a positive control, and vector without insert was used as a negative control. BPNP is a carboxylesterase inhibi-

TABLE I

Carboxyl Esterase Activities of Human and Mouse Egasyns Expressed in COS Cells Using pCD Vector Absorbance at 560 nm cDNA

2BPNP

1BPNP

Vector only Mouse egasyn (mEg) Human egasyn (hEg)

0.033 0.550 0.40

0.025 0.023 0.005

Note. Cells were lysed in 500 ml of 20 mM imidazole–HCl buffer, pH 7.4, by sonication. Fifty microliters of cell lysates in 780 ml of b-naphthyl acetate (0.32 mM) and 200 ml of Na 2HPO 4 (0.2 M, pH 7.0) were incubated at 39°C for 10 min with or without BPNP (5 mM). Then 200 ml of 10% SDS containing 1 mg of Fast Garnet GBC salt was added and incubated at room temperature and absorbance was measured at 560 nm. The results are averages of two independent experiments.

tor that has been used to characterize the esterase activity of mEg (1, 2, 5). Table I shows that transfection of COS cells with hEg cDNA in this vector resulted in an 11-fold increase in BPNP-inhibitable esterase activity in COS cell extracts over endogenous esterase activity. The carboxylesterase activities seen following expression of both hEg and mEg were completely inhibited by BPNP (Table I). The increases in esterase activity in the transfected cells were also documented by activity-stained bands in native gels of cell extracts. Human Eg (Fig. 1A), which migrates more slowly than mEg, appeared as two distinct bands while mouse egasyn shows greater heterogeneity appearing as six distinct bands. However, each appeared as a sharp single band on SDS– PAGE (Fig. 1B). To evaluate the extent to which glycosylation contributes to the apparent molecular weights, extracts of COS cells, transfected with either mEg or hEg and labeled with Tran 35S, were immunoprecipitated and analyzed by SDS–PAGE and fluorography before and after deglycosylation with PNGase F. Before deglycosylation, hEg (Fig. 1B, hEg/2PNGaseF) migrated faster than mEg, with apparent molecular weights of 62 and 66 kDa, respectively. The apparent molecular weights after deglycosylation were 60 and 61 kDa for hEg (hEg/1PNGaseF) and mEg (mEg/1PNGaseF), respectively. The human egasyn expressed in transfected COS cells was visualized by immunofluorescence staining using specific anti-hCE antibody (Fig. 2), which shows strong staining of transfected (Fig. 2B) and very little staining of nontransfected COS cells (Fig. 2A). The ER distribution of hEg was suggested by a reticular network extending from the nuclear membrane into the cytoplasm (29). A similar pattern was shown for mEg (20). However, the punctate peripheral staining sug-

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FIG. 1. (A) Carboxyl esterase activity of hEg expressed in COS-7 cells. COS cells were transfected with hEg cDNA in pJC119 vector, harvested 72 h after transfection in imidazole buffer, and analyzed in 6% nondenaturing polyacrylamide gels. For visualization of esterase activity, the gel was incubated with a-naphthyl acetate and Fast Blue BB. As a negative control, COS cells transfected with vector (pJC119) only, and as a positive control, COS cells transfected with mEg in pJC119, were also analyzed. (B) Immunoprecipitation of hEg and mEg from transfected COS cells by anti-hCE antibody. After 60 h of transfection, cells were labeled with Tran 35S-Label for 1 h, harvested, and immunoprecipitated with anti-hCE antibody (see Materials and Methods). Portions of both mEg and hEg were treated with peptide N-glycosidase F. The treated (1PNGaseF) and untreated (2PNGaseF) samples were analyzed by SDS–PAGE followed by fluorography.

gests that the overexpressed protein moves beyond the ER and extends at least to the Golgi network. Northern blot analysis showed that hEg is predominantly expressed in human liver (Fig. 3). Lesser signals are seen in mRNA from human heart and lung. A prolonged exposure showed that it is also expressed in kidney (not shown). In mouse, however, the mRNA levels detected by S1 nuclease assay in the kidney were similar to those seen in liver (21). Reprobing the MTN blot with b-actin control did not show any significant difference in the b-actin mRNA levels between the liver and kidney (not shown) suggesting that the difference in the message levels between the liver and kidney in humans is not due to the difference in RNA amount loaded. Human egasyn associates with human GUSB and reduces its secretion. To address the questions of whether hEg is a functional homologue of mEg and associates with hGUSB, we coexpressed hEg and hGUSB in COS cells. The conventional method to study the interaction between mEg and mGUSB utilizes native gels to separate slower migrating mEg– mGUSB complexes which contain one to four egasyn molecules from free mGUSB. However, hGUSB does not readily separate into defined microsomal and lysosomal components on native gels (2). As an alternate strategy to determine whether hEg interacts with hGUSB in cells coexpressing both proteins, we used chemical cross-linking and coimmunoprecipitation with hGUSB by anti-hGUSB antibody. When cross-

linking and immunoprecipitation were carried out on cell extracts transfected with hGUSB cDNA only (Fig. 4, lane 1), a prominent band of hGUSB and a barely detectable band, presumed to be resident monkey Eg, were seen. A clearly identifiable band corresponding to hEg was coimmunoprecipitated with hGUSB from extracts of cells transfected with both hGUSB and hEg cDNAs and cross-linked (lane 2). These results indicated that hEg associates with hGUSB in transfected COS cells. Lane 3 shows that hEg can be coimmunoprecipitated with hGUSB without cross-linking from the cell extracts expressing both proteins. Similarly, mEg can be coimmunoprecipitated with mGUSB from cell extracts expressing both proteins without crosslinking. The association of hGUSB with hEg appeared to be specific for hGUSB in that hEg did not coimmunoprecipitate with b-hexosaminidase B nor with carbonic anhydrase IV when either of these proteins was coexpressed with hEg (data not shown). We reasoned that if hEg binds hGUSB and sequesters some of hGUSB in the ER, the total amount of hGUSB secreted into the growth media should be reduced. Accordingly, we coexpressed hGUSB cDNA with pJC119 vector alone, antisense HEXB, or hEg cDNAs in the same pJC119 vector in COS cells and measured the percentage of total synthesized that was secreted into the media. The antisense HEXB was used to control the size of cotransfecting vectors. The sense HEXB insert was not used as expressed HEXB might compete with hGUSB for the mannose 6-phosphate

INTERACTION OF HUMAN EGASYN WITH GLUCURONIDASES

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FIG. 3. Tissue distribution of hEg. A multitissue Northern blot was hybridized with full-length cDNA of hEg labeled with random primer and hybridized at 68°C for 2 h.

FIG. 2. Immunofluorescent localization of hEg in endogenous and transfected COS-7 cells. COS cells, endogenous (A) or transfected with hEg (B), were grown on coverslips, fixed, permeabilized, treated with anti-hCE antibody, and then visualized by confocal fluorescence microscopy with fluorescein-conjugated goat anti-rabbit IgG as described under Materials and Methods.

receptor and lead to increased secretion of hGUSB. The results, summarized in Table II, show that 7–9% less hGUSB was secreted when it was coexpressed with hEg than when expressed with vector only or with antisense HEXB. These results are compatible with increased retention of hGUSB by hEg in the ER. Human Eg–GUSB complex formation does not involve interaction between the esterase active site and GUSB C terminus. To explore the involvement of the esterase activity of hEg in binding hGUSB, we used the inhibitor BPNP. To explore the requirement for the C terminus of hGUSB for binding hEg, we used the Cterminal truncation mutant, lacking the C-terminal 18 amino acids. Figure 5 shows that both full-length and

C-terminal truncated hGUSB associate with hEg (lanes 1 and 3). Treatment with 5 mM BPNP failed to release a significant amount of hEg from the immune complex into the supernatant (lanes 2 and 4). Incubation of the hGUSB– hEg complex with up to 50 mM concentrations of BPNP, or at higher temperature, also had no effect on the amount of hEg released (results not shown). As with hGUSB, mouse GUSB associates with hEg, but only very weakly. The association was unaffected by BPNP treatment (lanes 5 and 6). On the other hand, when the mGUSB–mEg complex was treated with 5 mM BPNP, a substantial amount of mEg was released (lanes 7 and 8), as previously reported. These results demonstrate that hEg interacts

FIG. 4. Coimmunoprecipitation of hGUSB and mGUSB with hEg or mEg. At 60 h following transfection, cells were labeled with Tran 35S-Label, harvested, sonicated, treated (lanes 1 and 2) or not treated (lanes 3 and 4) with cleavable cross-linker DSP, and immunoprecipitated with anti-hGUSB (lanes 1–3) or anti-mGUSB (lane 4) antibodies. The immunoprecipitates were analyzed on 7.5% SDS– PAGE gels under reducing conditions. Transfections were with hGUSB (lane 1), hGUSB 1 hEg (lanes 2 and 3), and mGUSB and mEg (lane 4).

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DISCUSSION

TABLE II

Human GUSB Activities Were Measured in Cell Extracts and Media of COS Cells Cotransfected with hGUSB cDNA and Vector, Antisense HEXB, or hEg cDNAs in Equal Amounts (5 mg each) cDNAs coexpressed with hGUSB Expt. 1 1V 1hEg Expt. 2 1V 1hEg Expt. 3 1AntiHEXB 1hEg

b-Glucuronidase activity: Units/mg cell proteins Cell extracts

Media

Total

% Secreted

410 609

117 108

527 717

22 15

1243 954

304 116

1547 1070

20 11

520 876

163 175

683 1051

24 17

Note. After 76 h of transfection, media were harvested, and cells were washed twice with cold PBS, harvested in 0.3% DOC, and assayed for b-glucuronidase activity using 4-methylumbelliferyl-bD-glucuronide. One unit of GUSB activity is equal to 1 nmol of umbelliferone produced in an hour.

with hGUSB, but show that the nature of the interaction of hEg with hGUSB is different than the interaction between mEg and mGUSB (18). Interaction of rat– human chimeric GUSB with hEg. In the course of our study we observed that rGUSB appears to interact with hEg, but only weakly compared to hGUSB. To locate the site in GUSB, which accounts for the greater interaction, we utilized a series of chimeric cDNAs constructed earlier from hGUSB and rGUSB cDNAs using conserved restriction sites (30). As shown in Fig. 6, the ratio of the signal of hEg coimmunoprecipitated with rGUSB (RBG) was only 33% as great as the ratio of the signal for hEg coimmunoprecipitated with hGUSB. The ratio of hEg signal to GUSB signal for the Rc1H chimera, which contains the N-terminal 228 amino acids from rat and the rest of the 652 amino acids from human, was comparable to that of hEg coimmunoprecipitated with whole hGUSB. Increasing the proportion of N-terminal rat amino acid sequences to amino acid 404 in chimera Rc2H decreased the ratio of coimmunoprecipitated hEg to chimeric GUSB to the level of hEg coimmunoprecipitated with rGUSB. When the chimeric GUSB (RHaR) contained only the 51 amino acids of human sequence encoded by the human cDNA between Cla I(683) and Afl II(838) and was composed of rat sequences otherwise, the ratio of coimmunoprecipitated hEg to chimeric GUSB increased twofold compared to the ratio of coimmunoprecipitated hEg to rGUSB, suggesting that these 51 amino acids (between 228 and 279) contain a site that contributes significantly to hEg binding by hGUSB.

The C-terminal sequences (usually KDEL or a closely related sequence) of ER luminal proteins provide the signal for their retention in the ER (26 –28). Presumably, the HIEL sequence in the C terminus of hEg provides the ER retention signal by which hEg is predominantly localized in the ER. Although mGUSB contains no ER retention signal, a substantial fraction of newly synthesized mGUSB is retained in the ER, at least in liver and kidney. Retention of mGUSB via binding to the accessory protein mEg in mice was shown more than 30 years ago. Although the existence of an egasyn-like molecule has been demonstrated in rat, rabbit, and frog, its presence in human tissues has not been demonstrated. The studies presented here show that an egasyn-like molecule does exist in human liver. Northern blot analysis (Fig. 3) clearly indicated its abundance in human liver and its presence in other tissues. However, unlike its mouse homologue, it is not abundant in kidney. Its sequence revealed it to be nearly identical to two previously reported sequences for human carboxyl esterase. The cloned hEg cDNA shares 78% homology with the mEg cDNA. The activesite residues, the Cys residues, and one of the potential glycosylation sites are conserved between the human and murine proteins and occur in regions of high similarity. Carboxyl esterases such as mEg and hEg are serine hydrolases that contain Ser and His residues at the active site. The conserved Ser is in a segment of 21 identical amino acids, and the conserved His is in a segment of 5 identical amino acids. The so-called “cat-

FIG. 5. Coimmunoprecipitation of egasyns with GUSBs and release of egasyns by BPNP treatment. At 60 h posttransfection with hGUSB or mGUSB cDNAs, and hEg or mEg cDNAs, cells were labeled, harvested, sonicated, and immunoprecipitated as in Fig. 4 with the respective anti-hGUSB or mGUSB antibodies. The washed, resuspended immunoprecipitates were treated with 5.0 mM BPNP and 5% 2-mercaptoethanol overnight at room temperature in the dark and resedimented, and the pellets (lanes 1, 3, 5, and 7) and supernatants (lanes 2, 4, 6, and 8) were analyzed on 7.5% SDS– PAGE gels and autoradiographed. The GUSB expressed and immunoprecipitated is indicated at the top. The cDNAs express full-length GUSB except lanes 3 and 4, which express truncated DhGUSB, missing the C-terminal 18 amino acids. The coexpressed Egs were hEg for lanes 1– 6 and mEg for lanes 7 and 8.

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FIG. 6. Coimmunoprecipitation of hEg with human, rat, and rat– human chimeric GUSBs. COS cells, cotransfected with an equal amount of hEg and one of different GUSB cDNAs, were labeled with Tran 35S-Label for 2 h at 60 h posttransfection, harvested, and immunoprecipitated with anti-hGUSB (HBG) or anti-rGUSB (RBG) or a mixture of both (all chimera) as in Fig. 4. After analysis by SDS–PAGE and fluorography, the lanes were scanned by a Scion gel plotter. The different constructs were hGUSB (HBG), rGUSB (RBG), RkH (59-KpnI site of the rat sequence replaced by the human sequence), Rc1H (59-ClaI at 683 of the rat sequence replaced by the human sequence), Rc2H (59-ClaI at 1211 of the rat sequence replaced by the human sequence), and RHaR (155 bp of human sequence between ClaI at 683 and AflII at 838 replaced the rat sequence). Values for hEg/GUSB ratios were obtained from the scanned value of hEg divided by the scanned value of GUSB in the respective lane. In these experiments hEg appeared as a doublet (probably corresponding to glycosylated and nonglycosylated forms) and both bands were included in the quantitation of hEg.

alytic triad” of serine proteases also includes an Asp near the His residue, but location of this Asp in mEg has not been established. Availability of the cDNAs for both hGUSB and hEg provided us with an opportunity to determine whether hEg binds hGUSB. Three lines of evidence indicate that hEg binds glucuronidase. First, with or without cross-linking, hEg is coimmunoprecipitated with antihGUSB antibody from cells coexpressing hEg and hGUSB (Fig. 4). Second, the converse is also true. Anti-hCE antibody can coimmunoprecipitate hGUSB from COS cells coexpressing hGUSB and hEg (not shown). Third, a band of the size expected for hEg can be precipitated with anti-hGUSB antibody from COS cells expressing hGUSB only, which suggests that endogenous monkey Eg binds hGUSB (Fig. 4, lane 1).

Also, endogenous COS cells were stained slightly with anti-hCE antibody (Fig. 2A). Several lines of evidence suggest that GUSB binding by egasyn in mouse and human tissues involves different mechanisms. First, the octapeptide (FGSRPFTF) in the C terminus of mGUSB has been found to be responsible for binding with mEg and is perfectly conserved in rat GUSB. However, it is not well conserved in hGUSB (LENSPFT). The C-terminal Phe is not present in hGUSB and the Arg between Ser and Pro is missing in hGUSB. Prior studies showed that replacement of Arg with Ile in mGUSB had no effect on complex formation with mEg (18). However, the role of terminal Phe is ambiguous: deletion led to loss of complex formation as assessed by native gels, but did not abolish egasyn-dependent

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ER retention (18). The Gly upstream of the serpinlike sequence is replaced by an Arg in W26 mice, which results in decreased ER retention and complex formation with mEg in those mice (31). This Gly is replaced by a Glu in hGUSB. Presumably these several differences in the C-terminal sequence of hGUSB make it no longer a substrate for the serine protease-like mEg. It has been shown experimentally that mEg does not bind monkey GUSB (20). Second, our results indicate that the C terminus is not required for binding of hGUSB by hEg. The truncated form of hGUSB (in which the last 18 amino acids were deleted) bound hEg (Fig. 5, lane 3). A non-C-terminal binding site is known for a cell surface receptor on hepatocytes, the serpin enzyme complex receptor, which recognizes an internal pentapeptide (FVFLM) sequence at residues 370 –374 of a1-AT in the a1-AT– elastase complex (32). The binding site on hGUSB for hEg has not yet been established. A third difference between murine and human Eg– GUSB complexes is that, in contrast to the mEg–mGUSB complex, the hEg– hGUSB complex was insensitive to BPNP treatment. BPNP is a member of a class of organophosphorus compounds which are potent inhibitors of serine proteases (4, 14). The inability to dissociate the hEg–GUSB complexes with BPNP treatment suggests that the interactions between hEg and GUSB are independent of the esterase active site of hEg, as has been observed in the case of the interaction between the C-reactive protein (CRP) and carboxyl esterases (33, 34). CRP is synthesized in hepatocytes and retained within the ER lumen by formation of complexes with two distinct but similar carboxyl esterases containing HXEL retention signals (35). CRP is secreted into plasma during the acute-phase response due to markedly reduced binding capacity of the ER carboxyl esterases (33). Another example of interaction between an esterase and other secretory proteins where the active site of esterase is not involved is the interaction between protective protein, a serine carboxyl peptidase, and b-galactosidase (36). These two proteins associate early in the biosynthetic pathway in the rough ER, but in this case the complex is not retained within the ER. In summary, our findings establish that a mEglike carboxyl esterase exists in human liver, and the recombinant enzyme binds hGUSB. However, in contrast to mEg, which recognizes a serpin-like sequence in the mGUSB C terminus and utilizes its esterase active site to form a complex, hEg appears to bind GUSB independently of its esterase active site. Also unlike the case with mEg binding mGUSB, the binding of hGUSB by hEg does not depend on the C-terminal 18 amino acids. On the other hand, the evidence suggests that an internal segment of 51

amino acids between 228 and 279 residues of hGUSB contributes to hEg binding. ACKNOWLEDGMENTS We thank Dr. Roger Ganschow of the Children’s Hospital Medical Center (Cincinnati, OH) for providing mouse egasyn cDNA, Dr. Frank Gonzalez of the National Cancer Institute (Bethesda, MD) for anti-hCE antibody, Dr. Richard Proia of the National Institutes of Health (NIDDK, Bethesda, MD) for hexosaminidase A cDNA, Dr. John Corbett for help in immunofluorosence, Dr. Yoshiro Nagao for help in cDNA cloning, and Ms. Elizabeth Torno for editorial assistance.

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