extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Liver Cytochrome P450 3A Endoplasmic Reticulum-associated Degradation

The CYP3A subfamily of hepatic cytochromes P450, being engaged in the metabolism and clearance of >50% of clinically relevant drugs, can significantly influence therapeutics and drug-drug interactions. Our characterization of CYP3A degradation has indicated that CYPs 3A incur ubiquitin-dependent proteasomal degradation (UPD) in an endoplasmic reticulum (ER)-associated degradation (ERAD) process. Cytochromes P450 are monotopic hemoproteins N-terminally anchored to the ER membrane with their protein bulk readily accessible to the cytosolic proteasome. Given this topology, it was unclear whether they would require the AAA-ATPase p97 chaperone complex that retrotranslocates/dislocates ubiquitinated ER-integral and luminal proteins into the cytosol for proteasomal delivery. To assess the in vivo relevance of this p97-CYP3A association, we used lentiviral shRNAs to silence p97 (80% mRNA and 90% protein knockdown relative to controls) in sandwich-cultured rat hepatocytes. This extensive hepatic p97 knockdown remarkably had no effect on cellular morphology, ER stress, and/or apoptosis, despite the well recognized strategic p97 roles in multiple important cellular processes. However, such hepatic p97 knockdown almost completely abrogated CYP3A extraction into the cytosol, resulting in a significant accumulation of parent and ubiquitinated CYP3A species that were firmly ER-tethered. Little detectable CYP3A accumulated in the cytosol, even after concomitant inhibition of proteasomal degradation, thereby documenting a major role of p97 in CYP3A extraction and delivery to the 26 S proteasome during its UPD/ERAD. Intriguingly, the accumulated parent CYP3A was functionally active, indicating that p97 can regulate physiological CYP3A content and thus influence its clinically relevant function.

Thus, consistent with a typical ERAD process, we have found that CYP3A ERAD involves posttranslational phosphorylation by cytosolic kinases (15,32,33); ubiquitination by the ER-integral "glycoprotein" 78/autocrine motility factor receptor (gp78/AMFR) and the cytosolic C terminus of Hsp70-interacting protein (CHIP) E3 Ub ligases along with their respective cognate Ub-conjugating enzymes UBC7/Ube2g2 and UbcH5a (33)(34)(35); and subsequent degradation by the 26 S proteasome (12)(13)(14)(15)(16)(17). As a monotopic ER protein, the bulk of the P450 molecule is exposed to the cytosol and thus amply accessible to the 26 S proteasome, a fraction of which is thought to be intimately associated with the ER membrane (36,37). Thus, it was unclear whether ER membrane extraction into the cytosol would be at all necessary for P450 proteasomal processing, as in the case of polytopic transmembrane or luminal ER proteins (21, 38 -40, 42-55). The latter require the p97 AAA ATPase-Npl4-Ufd1 chaperone complex for their retrotranslocation from the ER into the cytosol, a process that is fueled by ATP hydrolysis (21, 38 -40, 42-56).
p97, also known, albeit erroneously, as VCP (valosin-containing protein) or Cdc48p (in yeast), is an abundant cytosolic AAA ATPase (ATPase associated with various cellular activities) involved in an ever growing number of cellular functions and processes (55)(56)(57)(58)(59)(60)(61). Perhaps its best characterized functional role is its ER to cytosol retrotranslocation of ER integral and luminal proteins, a prerequisite for their subsequent UPD (21, 38 -40, 42-61). p97 has a homohexameric barrel structure with each subunit composed of two ATPase domains stacked on top of each other (62)(63)(64). It functions as a heterotrimeric complex with two additional heterodimeric adapters, Ufd1p and Npl4p, which bind to its N-terminal domain (49 -57). Ufd1p and Npl4p are thought to assist in the recruitment of polyubiquitinated target substrates to the p97 complex by engaging the poly-Ub chains decorating an ERAD target substrate (49 -51, 55-57).
Because Saccharomyces cerevisiae yeast strains with defects in each of the three individual components of the homologous Cdc48p-Npl4p-Ufd1p chaperone complex are available (21,40,42), we examined CYP3A4 ERAD in each of these defective strains along with the corresponding wild type strains upon heterologous expression of CYP3A4 (19). Indeed, findings of our stationary chase analyses revealed that CYP3A4 was markedly stabilized in the cdc48-2 yeast and even more strikingly so in the npl4-1 and ufd1-1 strains, relative to that in their wild type counterparts (19). These results provided the initial clue that despite its ER topology, strategically poised for imminent access to the 26 S proteasome, CYP3A4 ERAD in S. cerevisiae requires the yeast Cdc48p-Npl4p-Ufd1p chaperone complex. These findings led us to examine the role of p97 in cultured rat hepatocytes (14). Collectively, these findings provided evidence of a strong association between the native CYP3A and p97 that was significantly enhanced after mechanism-based CYP3A inactivation by the suicide inactivator, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP). DDEP, in the course of its CYP3A-catalyzed oxidation, results in the oxidative fragmentation of the CYP3A prosthetic heme into mono-and dipyrrolic products that irreversibly bind to the active site of the enzyme (11,65), fatally damaging it both structurally and functionally. In vivo, this structurally damaged protein with active site lesions within its cytosolic domain is marked for rapid clearance via ERAD/UPD in a typical ERAD-C process.
Given on one hand the imminent accessibility of CYPs 3A to the 26 S proteasome that renders p97 potentially dispensable in its ERAD and, on the other, this collective evidence in yeast and rat hepatocytes supporting a role for the p97 complex in CYP3A ERAD, we sought to determine the relative importance of the p97 complex to CYP3A ERAD/UPD through RNA interference (RNAi) targeted against p97. Our findings of p97 knockdown reveal that despite the predominantly cytosol-oriented ER topology of CYP3A, p97 plays a major role in CYP3A extraction into the cytosol and its subsequent delivery to the 26 S proteasome. Accordingly, p97 knockdown results in a strikingly marked accumulation of both the native and inactivated parent and ubiquitinated CYP3A species that remain firmly tethered to the ER membrane. Surprisingly, the native non-ubiquitinated CYP3A species accumulating in the ER on p97 knockdown is functionally active. This suggests that the p97 complex can regulate the hepatic content of the parent non-ubiquitinated CYP3A species and thus may critically influence its physiological function. Given the vast array of clinically relevant drugs, carcinogens, and other xenobiotics that depend on these particular P450s for their metabolic clearance (1), our findings, if extrapolated to the human liver, would be clinically and/or toxicologically relevant.
Isolation of Microsomes and Cytosol from DDEP-treated Rats-Male Sprague-Dawley rats (6 -8 weeks old) purchased from Simonsen Laboratories (Gilroy, CA) were fed and given water ad libitum and handled according to institutional animal care and use committee guidelines. After acclimation for ϳ5-7 days, they were pretreated with the CYP3A inducer Dex (100 mg/kg/day; i.p.) in corn oil for 4 days. Twenty-four h after the last dose, they were given a single i.p. injection of DDEP (125 mg/kg) and killed 2 h later. Livers were perfused and isolated, and homogenates were prepared and subjected to ultracentrifugation at 100,000 ϫ g at 4°C for 1 h; and microsomes (100,000 ϫ g pellet) and/or cytosol (100,000 ϫ g supernatant) were prepared as described previously (11)(12)(13). Dex-pretreated rats not given DDEP were killed at time 0 h and used as the corresponding controls. Microsomes were washed free of cytosol by two sequential rounds of resedimentation at 100,000 ϫ g at 4°C for 30 min and resuspension, and stored as "pellets" overlaid with Tris buffer, pH 7.4, 20% (v/v) glycerol, EDTA (1 mM) at Ϫ80°C until use.
Lentiviral Packaging in HEK 293T Cells-This was carried out exactly as described (35). A 5-10-ml aliquot of the filtered viral stock was used for total RNA extraction. The extracted RNA concentration was determined by the nanodrop technique (average concentration is 100 ng/l). The samples were stored at Ϫ80°C until the reverse transcription step to generate cDNA. The lentiviral RNA concentration was also determined by real-time quantitative PCR (qRT-PCR) analyses (68) using the following: forward primer (HIV1-LTR-FO), 5Ј-TGTGTGCCCGTCTGTTGTGT-3Ј; reverse primer (HIV1-LTR-RE), 5Ј-GAGTCCTGCGTCGAGAGAGC-3Ј; and the Taqman probe (HIV1-LTR-TaqProbe), 5Ј-FAM-CAGTG-GCGCCCGAACAGGGA-TAMRA-3Ј (FAM, 6-carboxyfluorescein; TAMRA, tetramethyl rhodamine carboxylic acid derivative). Based on the RNA concentration of this 5-10-ml aliquot, the entire viral stock was pelleted and resuspended in PBS/hepatocyte culture medium to a final concentration of 1 g of viral RNA/ml for infection of hepatocytes as described (35).
Rat Hepatocyte Viral Infection and Culture-Hepatocytes were isolated from rats (4 -6 weeks old) by in situ liver perfusion with collagenase (liver digest medium) and cultured as described (14,66,67,69). Cells were maintained for 2 days with a daily change of medium to enable recovery and hepatic function restoration (70). At 72 h of culture, puromycin (5 g/ml) was added to the medium for selection of cells that were successfully infected by the shRNA-containing virus. The medium was replaced daily, the Dex concentration was increased to 10 M to induce CYPs 3A, and cells were cultured for an additional 5-6 days, with daily light microscopic examination for any signs of cell death or cytotoxicity. In some cases, cells were treated with vehicle (DMSO), Dex (10 M), or Dex (10 M) plus DDEP (100 M) for 4 h.
In preliminary experiments, the optimal time for Ն80% knockdown of p97 was determined to be Ͼ7 days. At this time, cells were harvested in lysis buffer (66), and clarified lysate supernatants were subjected to Western immunoblotting analyses. In parallel, some shRNA-infected cell cultures were also harvested in RNA-stabilizing reagent (Qiagen) and used for total RNA extraction and qRT-PCR analyses (see below).
Preparation of Microsomal and Cytosolic Fractions-Microsomes were isolated from rat hepatocytes as described (13,69). The supernatant obtained after the first 100,000 ϫ g ultracentrifugation was used as the cytosolic fraction and subjected to CYP3A immunoprecipitation as described below. To determine the content of CYP3A normally ER-integrated relative to that dislocated but still loosely associated with the external ER surface, microsomes from 35 S-pulse-chase experiments were subjected to a 0.1 M Na 2 CO 3 wash exactly as detailed (35). The 0.1 M Na 2 CO 3 -washed microsomes representing firmly integrated proteins were sedimented at 180,000 ϫ g, and the pellet was used for CYP3A immunoprecipitation. The resulting supernatant was precipitated with 100% trichloroacetic acid, and the protein pellet was washed with 100% acetone and referred to as the "Na 2 CO 3 -wash." This Na 2 CO 3wash was subjected to CYP3A immunoprecipitation as described below. It represents any proteins that were dislocated from the ER but remained loosely associated with the ER membranes.
CYP3A Immunoprecipitation-CYP3A was routinely immunoprecipitated before the extent of its ubiquitination or 35 S-incorporation was determined as described previously (14). For CYP3A immunoprecipitation, 1 mg of lysate protein or 2 mg of cytosolic protein was immunoprecipitated with 2 mg of goat anti-CYP3A antibody, whereas 500 g of microsomal protein was immunoprecipitated with 2 mg of goat anti-CYP3A antibody. The Na 2 CO 3 -washes (200 g of protein) were resuspended in immunoprecipitation buffer, boiled, and used for CYP3A immunoprecipitation with 500 g of goat anti-CYP3A antibody.
Immunoblotting Analyses-CYP3A and ubiquitinated CYP3A immunoprecipitates and ubiquitinated hepatic protein were immunoblotted as described except that 5% (w/v) nonfat milk in 0.1% (v/v) Tween TBS (TTBS) was used for blocking (17,66). Because the CYP3A antigenicity of the peptide-derived antibodies varied, the protein amounts loaded in each immunoblotting analyses also varied. Thus, with polyclonal ␣-Internal, ␣-C-terminal, and ␣-CYP3A IgGs, 20, 10, and 5 g of microsomal protein were loaded, respectively. For immunoblotting analyses of the cytosolic fraction, 20 g (␣-Internal IgG) and 10 g (polyclonal ␣-C-terminal and ␣-CYP3A IgGs) of protein were loaded. For p97 immunoblotting analyses, lysate protein (20 g) was subjected to 7.5% SDS-PAGE and electroblotted onto a nitrocellulose membrane, and the p97 protein was immunoblotted with a primary rabbit polyclonal antibody (sc-20799, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by goat anti-rabbit HRP-conjugated secondary antibody (catalog no. 170-6515) from Bio-Rad. All immunoblots were developed with the SuperSignal West maximum sensitivity Femto or Pico chemiluminescent substrate from Pierce. Actin immunoblotting analyses were routinely conducted with each lysate (10 g of protein) to ensure equivalent protein loading.
The beads were packed into small columns onto an Eppendorf tube containing 100 l of 1 M Tris-HCl, pH 6.8, for collection of the eluate. Bound antigen was eluted with 50 l of 0.1 M diethylamine, pH 11.5, containing 0.5% (w/v) DOC and 10% (v/v) glycerol. This elution step was repeated with an additional 50 l. Contents of the tube were mixed vigorously to adjust the pH to 7.4. Following this, the beads were repeatedly washed with borate buffer and stored in 0.1 M borate buffer with 0.02% (w/v) sodium azide at 4°C. After elution, these samples were used for Western immunoblotting analyses with goat anti-CYP3A23 antibody.
Densitometric Quantification-Direct quantification of the immunoblots was performed by ImageJ (National Institutes of Health) analyses as described (67). Densitometrically derived arbitrary units of individual immunoblots were normalized against corresponding values of the actin loading controls and then expressed as a percentage of the control/basal values. When [ 35 S]CYP3A immunoprecipitates were quantified, gels were dried, exposed to PhosphorImager screens, visualized using a Typhoon scanner, and quantified using ImageQuant software.
qRT-PCR Analyses-Total RNA was extracted and reverse transcribed to cDNA exactly as described (67,69). Universal PCR master mix (catalog no. 430447) and Taqman primerprobe mixes were purchased from Applied Biosystems Inc. (Foster City, CA) for the detection of rat mRNA sequences for p97 (catalog no. Rn00587865_m1), cytochrome b 5 (catalog no. Rn01483963_m1), CYP3A23 (catalog no. Rn01412959_g1), ␤-glucuronidase (catalog no. Rn00566655_m1), Grp78 (catalog no. Rn00565250_m1), Grp94 (catalog no. Rn01760569_m1), gp78, CHIP, and GAPDH (catalog no. Rn01775763_g1). The gp78 primers were ACCTCATGCAC-CACATTCACATGC (forward) and CAAGACACCTCTT-GTCCAACATGC (reverse). The CHIP primers were ACCCGGAACCCACTTGTGGCAGTG (forward) and CTG-GATGGGCAGTCTGTGAAGGCG (reverse). qRT-PCR was carried out as described (35). The Ct (cycle number at which the fluorescent signal reaches the threshold level) value reflecting the expression of each gene was normalized to that of the endogenous control glucuronidase gene. Relative gene expression was calculated as 2 Ϫ⌬Ct , where ⌬Ct is defined as Ct for the gene of interest minus Ct for glucuronidase. All values are expressed as percentage increase/decrease with respect to the corresponding RNA value in control shRNAinfected hepatocytes.
Immunofluorescence Microscopy-Immunochemically detectable CYP3A content of shRNA-infected and control cells was detected with a Zeiss Axiovert 200M, LSM 510 Meta confocal microscope as described previously (35). The relative immunofluorescence intensity was quantitated as the mean intensity of a cytoplasmic area (4 m ϫ 5 m) excluding the nucleus within each cell using the dedicated Zeiss confocal software. Intensity values (mean Ϯ S.D.; n ϭ 50 cells) ranged from 1 to 256, corresponding to the fluorescence dynamic range of 1 (no signal) to 256 maximal (saturation) immunofluorescence signal, respectively, of the 20 m 2 area. The relative nuclear diameters of control shRNA-infected and p97 shRNA 1ϩ2-infected hepatocytes (n ϭ 33 each) were determined as an index of apoptotic shrinkage induced by p97 knockdown.
CYP3A Functional Assay-A fluorescence-based P450 assay (72) was performed as described (35) by direct incubation of rat hepatocytes cultured in 60-mm Permanox plates and infected with p97 shRNA 1ϩ2 or control shRNA for 7 days. Results were expressed as mean Ϯ S.D. mol of HFC metabolite formed/3.5 ϫ 10 6 hepatocytes/h of three individual cultures.
Assay of Proteasomal Function-The Proteasome-Glo cellbased assay system was used to monitor the chymotrypsinlike, trypsin-like, and postglutamyl peptide hydrolytic (caspase-like) 20 S proteasomal core proteolytic activities (73) in intact rat hepatocytes with and without p97 knockdown to assess whether p97 RNAi had concomitantly affected hepatic proteasomal function. In brief, Suc-LLVY aminoluciferin, Suc-benzyloxycarbonyl-LRR aminoluciferin, or Suc-benzyloxycarbonyl-nLPnLD (where nL represents norleucine) aminoluciferin substrate probe dissolved in a cell-permeabilizing buffer was incubated with 20,000 intact rat hepatocytes for 10 min at room temperature. The aminoluciferin generated from each of these substrates following its release by the proteasomal chymotrypsin-like, trypsin-like, or caspase-like proteolytic activities, respectively, was monitored by an indirect coupled assay, wherein a recombinant luciferase utilizes the aminoluciferin produced to generate a luminescent signal (73). The luminescence was monitored using a Spectramax M5E plate reader in a 96-well plate assay. In parallel, some cells were incubated with the proteasomal inhibitor epoxomy-p97-mediated CYP3A ER Extraction into the Cytosol cin (10 M) or MG132 (50 M) and the lysosomal inhibitor NH 4 Cl (20 mM) for 3 h in order to verify the relative proteasomal versus lysosomal specificity of these proteolytic activities (supplemental Fig. S1).
Statistical Analyses-Experiments were performed in triplicate. The Kolmogorov-Smirnov test was performed on the data sets to ensure that the data were normally distributed. Data were compared by analysis of variance, and p values Ͻ0.05 were considered statistically significant.

CYP3A Extraction into the Cytosol of DDEP-treated Rats-
In preliminary experiments in rats treated with the CYP3A suicide inactivator DDEP for 0, 0.5, 1, and 2 h, we sought to define the time course of the ER extraction into the cytosol (data not shown). These initial findings along with our previously published observations in DDEP-treated rats and DDEP-incubated rat hepatocytes in suspension (12,13) revealed that, as expected of an ER membrane-anchored monotopic protein, a major fraction of hepatic CYP3A (CYP3A23/ CYP3A2) was initially (0 h) in the microsomal fraction, with a small amount in the cytosol. However, within 2 h of DDEP inactivation, CYP3A was largely lost from the ER and could be retrieved as the parent protein (55 kDa) and/or its high molecular mass (HMM; 65-250 kDa) ubiquitinated species in the cytosol. These cytosolic CYP3A species accumulated in the presence of proteasomal inhibitors (12)(13)(14). For these reasons, we used this 2-h period to scrutinize the process of CYP3A extraction from the ER. Was CYP3A extracted out as the intact protein, or was it cleaved from its ER membrane signal anchor and then delivered to the cytosolic 26 S proteasome?
Immunoblotting analyses with antibodies to the N-terminal proline-rich domain peptide (␣-Internal) and to the C-terminal peptide (␣-C-terminal) revealed that the protein spanning residues 35-502 initially existed largely in the liver ER membrane (microsomes) but was removed intact from the ER membrane into the cytosol over the 2 h of DDEP treatment (Fig. 1A). Similar findings were obtained in cultured rat hepatocytes treated with DDEP (100 M) for 4 h (data not shown). Parallel immunoblotting analyses of liver microsomal and cytosolic subfractions from these DDEP-treated rats with antibodies to the signal anchor (␣-NT) and basic sequence (␣-Middle) were unsuccessful because the antibodies recognized several N termini from other microsomal proteins and were nonspecific. We therefore resorted to immunoaffinity capture, wherein the ␣-NT was cross-linked to Protein A-Sepharose and then exposed to solubilized liver microsomes or cytosol from rats treated with DDEP for 0 -2 h. The ␣-NT immunoaffinity-captured fractions were then subjected to immunoblotting analyses with goat polyclonal anti-CYP3A antibody (Fig. 1B). These findings revealed that the CYP3A23 N terminus was indeed initially in the liver microsomes but progressively moved into the cytosol along with the rest of the protein over the 2-h period of DDEP treatment (Fig. 1B).
Corresponding Ub-immunoblotting analyses of CYP3A immunoprecipitates from liver microsomal and cytosolic subfractions from these same rats with goat polyclonal anti-CYP3A IgGs revealed CYP3A ubiquitination at 1 h, while the CYP3A was apparently largely in the ER, with the gradual loss of this ER-bound ubiquitinated species into the cytosol over the next 1 h (Fig. 1C). These findings are entirely consistent with our previous observations that upon inactivation, CYP3A is ubiquitinated while still in the ER, and subsequently extracted into the cytosol for proteasomal degradation (12)(13)(14). Our present findings additionally reveal that CYP3A is indeed dislocated intact from the ER membrane along with its extreme N-terminal signal anchor.
RNAi Evidence for an in Vivo Role of p97 AAA-ATPase Complex in Hepatic CYP3A Extraction-Given our previous findings both in yeast and cultured rat hepatocytes suggesting a plausible role for the p97 complex, we examined whether p97 was indeed involved in CYP3A extraction into the cytosol through RNAi directed against p97 (Fig. 2). Lentivirus-mediated delivery of three shRNAs targeted against the rat p97 FIGURE 1. Extraction of DDEP-inactivated CYP3A into cytosol. A, microsomes or cytosol from Dex-pretreated rats (n ϭ 3) treated with DDEP for 0 or 2 h were subjected to immunoblotting analyses with polyclonal antibody against the proline-rich (Internal), C-terminal, or intact CYP3A23 domains as the primary antibody, and the CYP3A content was densitometrically quantified as described (see "Experimental Procedures"). Corresponding actin loading controls are also included. A representative CYP3A immunoblot obtained with each antibody is shown at the top, and the corresponding densitometric quantification of three individual immunoblots is shown at the bottom. Values are mean Ϯ S.D. (error bars) of immunochemically detected CYP3A content in liver microsomes or cytosol from three individual rats, expressed as a percentage of their initial (0 h) values. Statistically significant differences in CYP3A content were observed between the two mean Ϯ S.D. values, each marked with the same symbol as follows. , p Ͻ 0.0001; ¶, p Ͻ 0.0001; †, p Ͻ 0.0001; §, p Ͻ 0.005; ‡, p Ͻ 0.001; F, p Ͻ 0.001. B, a typical CYP3A immunoblotting analysis with a goat polyclonal antibody against the intact protein is shown of CYP3A immunoaffinity-captured with an antibody against the extreme N terminus of the CYP3A23 signal anchor using the corresponding microsomes and cytosol shown in A. C, Ub-immunoblotting analyses of CYP3A immunoprecipitates from liver microsomes and cytosol obtained from rats treated with DDEP for 0, 1, or 2 h. A typical immunoblot developed with alkaline phosphatase-conjugated secondary antibody is shown. FEBRUARY 4, 2011 • VOLUME 286 • NUMBER 5 gene were used singly or in combination in cultured rat hepatocytes pretreated with Dex to induce CYP3A content and thus improve its detection ( Fig. 2A). A "control" shRNA was used in parallel as the corresponding vector control. qRT-PCR analyses of total RNA from hepatocytes infected with the control or p97-targeted shRNAs indicated that although all three shRNAs were capable of knocking down p97 mRNA and protein expression, the combination of shRNA 1ϩ2 seemed to be the most effective, with a knockdown of Ͼ85% p97 mRNA expression ( Fig. 2A), and Ͼ90% p97 protein expression (Fig. 2B). This particular shRNA 1ϩ2 combination was therefore used in all subsequent studies. Parallel qRT-PCR analyses of shRNA 1ϩ2-infected hepatocytes indicated no corresponding mRNA knockdown of CYP3A23, cytochrome b 5 , and gp78 (three integral ER proteins); Grp78/BiP and Grp94 (two ER-associated chaperones); CHIP (a cytosolic E3 Ub ligase cochaperone); or GAPDH and glucuronidase (two housekeeping genes), thereby attesting to the specificity of the p97 knockdown (Fig. 2C). Additionally, the findings of essentially unaltered hepatic Grp78/BiP and Grp94 mRNA levels, sensitive indices of ER stress induction/unfolded protein response, also attest to no such intracellular perturbation upon such an extensive p97 knockdown.

p97-mediated CYP3A ER Extraction into the Cytosol
Corresponding immunoblotting analyses of hepatocyte lysates also revealed a marked Ͼ4.5-fold stabilization of the native parent CYP3A protein (55 kDa) over the control levels in hepatocytes infected with shRNA 1ϩ2, versus any other combination or any of the individual shRNAs (Fig. 3A). However, the individual shRNAs also significantly stabilized CYP3A content over the control levels, and the shRNA 2ϩ3 combination led to a respectable and statistically significant 3.5-fold stabilization of the native CYP3A protein over the control levels (Fig. 3A). Ub-immunoblotting analyses of CYP3A immunoprecipitates from lysates of cells infected with shRNA 1ϩ2 showed a marked accumulation of HMM ubiquitinated CYP3A species, which was further enhanced upon DDEP-induced CYP3A inactivation (Fig. 3B). These findings unequivocally indicated that p97 knockdown indeed stabilized the hepatic content of both the parent and ubiquitinated CYP3A species.
In situ confocal immunofluorescence microscopic analyses of hepatocytes infected with the control shRNA or p97 shRNA 1ϩ2 showed no significant effects on cellular morphology following 7 days of infection. Thus, no deleterious

p97-mediated CYP3A ER Extraction into the Cytosol
cellular effects and no evidence of apoptotic morphology or nuclear hypertrophy and/or other perturbations characteristic of apoptosis upon such an extensive (Ͼ90%) endogenous p97 protein knockdown were detected. In fact, the nuclear diameter (14.6 Ϯ 1.44 m; n ϭ 33 cells) of the p97 knocked down hepatocytes was found to be comparable to that of the corresponding control shRNA-infected cells (14.0 Ϯ 1.41 m; n ϭ 33 cells). By contrast, the immunofluorescent CYP3A content, consistent with the immunoblotting analyses, was visibly increased in p97 shRNA 1ϩ2-infected cells over the controls (Fig. 3C). Accordingly, quantification of the relative immunofluorescence intensity of a 20-m 2 cytoplasmic area (excluding the nucleus) of control and p97 shRNA 1ϩ2-infected cells yielded values of 47.2 Ϯ 4.8 and 116 Ϯ 6.3 (mean Ϯ S.D.; n ϭ 50 cells), respectively.
To determine whether the stabilized hepatic CYP3A fraction represented a functional CYP3A species or a structurally inactive species already marked for cellular disposal, we examined the functional activity of the CYP3A stabilized after p97 knockdown in cultured rat hepatocytes "in vivo" (Fig. 3D).
We therefore monitored the CYP3A-dependent O-debenzylation of BFC, a relatively selective diagnostic probe for CYP3A function, in a fluorescence-based assay (72). A significant increase of HFC, the O-debenzylated BFC metabolite, was detected in the extracellular medium after p97 knockdown relative to corresponding controls at 1 h (Fig. 3D). No corresponding increase in HFC formation was detected in the cell lysates, thereby revealing that most of the HFC formed was exported into the extracellular medium. These findings in intact non-DDEP-treated hepatocytes indicate that the CYP3A stabilization observed upon p97 knockdown is functionally relevant.
A Definitive Role for p97 in the ER Extraction of Hepatic CYP3A-A considerable fraction of the 26 S proteasome is associated with the ER membranes (36,37), and the relative extent to which the p97 complex and the 26 S proteasome are involved in the extraction of a monotopic ERAD-C substrate, such as hepatic CYP3A, was unclear. Liver microsomal and cytosolic subfractions from control shRNA and shRNA 1ϩ2infected hepatocytes in culture were therefore examined with Ϫp97 cells were treated with the CYP3A suicide inactivator DDEP for 4 h. CYP3A immunoprecipitates (IP) from lysates derived from DDEP-treated control and Ϫp97 cells at 0 and 4 h were subjected to Ub-immunoblotting analyses (IB) as detailed (see "Experimental Procedures"). A representative immunoblot is shown. C, in situ verification of CYP3A stabilization in cultured rat hepatocytes with confocal immunofluorescence microscopy. Shown are rat hepatocyte cultures infected with the control shRNA (Control), or p97 shRNA 1ϩ2 (Ϫp97) for 7 days. On the 8th day, shRNA-infected rat hepatocyte cultures were fixed and stained with antibodies to CYP3A (green). Data from a representative experiment showing relative CYP3A accumulation are shown. D, functional relevance of hepatic CYP3A stabilization after p97 knockdown (Ϫp97). Rat hepatocyte cultures were infected with the control shRNA or p97 shRNA 1ϩ2 (Ϫp97) for 7 days. On the 8th day, CYP3A functional activity was assayed in intact hepatocytes by assessing their ability to catalyze the 7-O-debenzylation of BFC, a diagnostic CYP3A functional probe, to HFC. The relative HFC formation (mol of HFC formed/3.5 ϫ 10 6 cells/h) in the medium was assayed (as detailed under "Experimental Procedures"). Experimental values (mean Ϯ S.D.) from three individual experiments are shown. Statistical analyses revealed significant differences in hepatic CYP3A function between control and Ϫp97 cells at p Ͻ 0.01. FEBRUARY 4, 2011 • VOLUME 286 • NUMBER 5 or without DDEP treatment (Fig. 4A). In the absence of DDEP treatment, immunoblotting analyses revealed that most of the native CYP3A is in the microsomes, with a small fraction in the cytosol. However, following DDEP treatment for 4 h, CYP3A immunoblotting analyses of microsomes derived from these cells documented the characteristic loss of parent CYP3A protein (ϳ55 kDa), stemming from its DDEP-induced UPD/ERAD (Fig. 4A). CYP3A immunoblotting analyses of the cytosolic subfraction isolated from these same cells showed a corresponding increase of CYP3A protein, consistent with the loss of ER-bound CYP3A protein species upon its dislocation/ extraction from the ER membrane into the cytosol (Fig. 4A). p97 knockdown markedly increased not only the native CYP3A microsomal content ( Fig. 4A; ϪDDEP, lane 3) but also that of the DDEP-inactivated CYP3A species (Fig. 4A;  ϩDDEP, lane 4). By contrast, p97 knockdown significantly abrogated the appearance of any native or DDEP-inactivated parent CYP3A ϳ55-kDa species into the cytosol (Fig. 4A,  lanes 7 and 8). Corresponding densitometric quantification of the relative CYP3A content after immunoblotting analyses is shown (Fig. 4A, bottom) and supports this assessment. This provided the first clear evidence that p97 was indeed crucial for CYP3A dislocation from the ER.

p97-mediated CYP3A ER Extraction into the Cytosol
However, it was plausible that the CYP3A species accumulating in the cytosol following p97 knockdown was largely ubiquitinated and thus at higher molecular masses not reflected by the parent CYP3A 55 kDa band. We therefore subjected CYP3A immunoprecipitates from the same microsomal and cytosolic subfractions examined in Fig. 4A to Ub-immunoblotting analyses (Fig. 4B). These findings revealed that indeed, in cells infected just with the control shRNA and no other treatment, a considerable fraction of CYP3A was found ubiquitinated both in the microsomes and cytosol (Fig. 4B). DDEP treatment, as expected, further increased the extent of this ubiquitination in both cellular compartments (Fig. 4B). However, upon p97 knockdown, the fraction of both the ubiquitinated native and DDEP-inactivated HMM CYP3A species dramatically increased in the microsomes while visibly declining in the cytosol (Fig. 4B). Together, these findings are entirely consistent not only with a major role for p97 in the dislocation of the parent as well as the ubiquitinated CYP3A species into the cytosol, but also with the ubiquitination of CYP3A occurring to a substantial degree while the protein is still in the ER by the ER-integral Ub ligase gp78 and/or cytosolic CHIP (33)(34)(35).
p97 Rather than the 26 S Proteasome Plays a Major Role in the ER Extraction of Hepatic CYP3A-The above findings were surprising because hepatocytes with p97 specifically knocked down still retained their full 26 S proteasomal complement. Indeed, a cell-based bioluminescent assay monitoring three proteasomal ATPase-independent proteolytic activities (trypsin-like, chymotrypsin-like, and caspase-like, corresponding to 20 S proteasomal ␤2, ␤5, and ␤1 subunits (73)) indicated no significant differences in proteasomal function between the control shRNA-infected cells and p97 shRNA 1ϩ2-infected cells ( Fig. 5A and supplemental Fig. S1). Thus, the functionally intact 26 S proteasome could have compensated for such a major loss of p97 function by both dislocating the ubiquitinated CYP3A and degrading it. As such, if all of the HMM ubiquitinated CYP3A species extracted by the 26 S proteasome into the cytosol were to be promptly degraded by it, it would be difficult to assess the specific contribution of p97 relative to that of the 26 S proteasome to the ER extraction of ubiquitinated CYP3A. We therefore examined the effect of blocking such proteasomal degradation were it to occur, by concomitant treatment of the cells with MG262, an effective inhibitor of 20 S proteasomal Thr proteases (74). Cultured hepatocytes were infected with either the control shRNA or with p97 shRNA 1ϩ2 for 7 days, as detailed above. On the 8th day, they were treated at time 0 with MG262 (10 M), followed 1 h later by DDEP. After 4 h of DDEP treatment, control and MG262-treated cells were harvested, and microsomal and cytosolic subfractions were subjected to CYP3A immunoprecipitation (see "Experimental

p97-mediated CYP3A ER Extraction into the Cytosol
Procedures") (Fig. 5B). Ub-immunoblotting analyses of CYP3A immunoprecipitates derived from the microsomal and cytosolic subfractions from DDEP-treated control cells once again exhibited the pronounced CYP3A ubiquitination seen in Fig. 4B, consistent with DDEP-mediated enhancement of CYP3A ERAD/UPD (Fig. 5B). Furthermore, as also documented in Fig. 4B, the dislocation of HMM ubiquitinated CYP3A protein species into the cytosol was markedly diminished upon p97 knockdown (Fig. 5B). However, when the hepatic proteasomal (proteolytic) function was concomitantly inhibited by MG262 treatment of hepatocytes, the level of ubiquitinated CYP3A species in the cytosol upon p97 knockdown did not significantly increase. In fact, it remained considerably lower than that in the cytosol of corresponding DDEP-treated control cells containing a functional p97 (Fig.  5B). On the other hand, the level of HMM ubiquitinated CYP3A species in the microsomal fraction not only increased upon p97 knockdown, consistent with the findings depicted in Fig. 4B, but CYP3A ubiquitination also was found to extend to an even higher molecular mass range with the concomitant MG262-mediated proteasomal inhibition (Fig. 5B). These findings thus reveal not only that p97 rather than the 26 S proteasome plays a major role in CYP3A dislocation but also that, upon concomitant inhibition of the proteasomal function, the CYP3A species accumulating in the ER, undergoes further ubiquitination to higher molecular mass species.
CYP3A Species Accumulating in the ER upon p97 Knockdown Is Largely Anchored to the ER-CYPs 3A, in common with most hepatic microsomal P450s, are highly hydrophobic proteins that, even after cleavage from their ER membrane anchor, tend to partition into the microsomal subfraction upon ultracentrifugation (4,5). We therefore examined the relative extent of CYP3A (i) truly anchored to the ER membrane and thus not removed by a 0.1 M Na 2 CO 3 wash of the liver microsomes; (ii) solubilized by the 0.1 M Na 2 CO 3 wash of the liver microsomes and thus representing the CYP3A species dislocated from the ER membrane but still loosely associated with its external surface; and (iii) in the cytosolic subfraction representing the CYP3A species dislocated from the ER membrane and truly extracted into the cytosol (Fig. 6A). To enhance CYP3A detection, the control shRNA-infected and shRNA 1ϩ2-infected cells were first pulse-chased with [ 35 S]Met/Cys for 1 h and then, following chase with unlabeled Met/Cys, treated for 4 h with DDEP. Cells were then harvested, and the various organellar subfractions were prepared. [ 35 S]CYP3A immunoprecipitated from each of these subfractions was subjected to SDS-PAGE and PhosphorImager scanning and ImageQuant analyses of the dried gels as detailed (see "Experimental Procedures") (Fig. 6A).
These combined analyses of [ 35 S]CYP3A immunoprecipitates revealed that, in untreated control shRNA-infected hepatocytes, [ 35 S]CYP3A is initially largely present as an integral 55-kDa ER protein with minimal ubiquitination detectable as the HMM species ranging between 65 and 250 kDa (Fig.  6A, top). A smaller fraction that includes both parent (55 kDa) and HMM ubiquitinated [ 35 S]CYP3A species was found in the cytosol, and an even smaller fraction was found to be dislocated from the ER but loosely bound to it. The latter fraction recovered in the 0.1 M Na 2 CO 3 microsomal wash was trichloroacetic acid-precipitable and immunoprecipitable. However, after 4 h of DDEP treatment, the ER-integral parent [ 35 S]CYP3A species rapidly declined, whereas the corresponding HMM [ 35 S]CYP3A species increased, consistent with DDEP-mediated structural inactivation and subsequent ubiquitination of the protein while still anchored to the ER membrane (Fig. 6A, top). CYP3A immunoprecipitation analyses of the corresponding cytosolic subfraction also revealed some parent species but an increase of the ubiquitinated [ 35 S]CYP3A species, consistent with a substantial fraction of the DDEP-inactivated P450 protein being extracted into the FIGURE 5. Relative contribution of p97 versus the 26 S proteasome to the extraction of ubiquitinated CYP3A from the ER. A, assessment of the effects of p97 knockdown on hepatic proteasomal function. Cells were infected with control shRNA or p97 shRNA 1ϩ2 (Ϫp97) for 7 days, and on the 8th day they were examined with the specific probes for the proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities in a cell-based bioluminescent assay as detailed (see "Experimental Procedures"). Values are mean Ϯ S.D. (error bars) relative luminescence units derived from three separate Ϫp97 cultures and expressed as a percentage of the values for corresponding activities in control shRNA-treated cells. No statistically significant differences were observed between activities from control and Ϫp97 cells. B, cells were infected with control shRNA or p97 shRNA 1ϩ2 (Ϫp97) for 7 days and then on the 8th day treated with or without DDEP for 4 h as described in the legend to Fig. 4. Some DDEP-treated cells were also treated concomitantly with the proteasomal inhibitor, MG262. Microsomes or cytosol were isolated and subjected to CYP3A immunoprecipitation. The corresponding CYP3A immunoprecipitates from microsomes or cytosol were subjected to Ub-immunoblotting analyses and development with alkaline phosphatase-conjugated secondary antibody as detailed (see "Experimental Procedures"). A representative immunoblot is shown. cytosol in the presence of a functional p97 (Fig. 6A, top). The Na 2 CO 3 wash after 4 h of DDEP treatment revealed no appreciable increase in [ 35 S]CYP3A fraction that remained ER-associated upon dislocation (Fig. 6A, top). These findings confirm our previous findings that DDEP-mediated CYP3A inactivation enhances its ubiquitination and dislocation from the ER membrane into the cytosol, wherein it is normally degraded by the 26 S proteasome (13).
On the other hand, following p97 knockdown of untreated hepatocytes, the parent [ 35 S]CYP3A species firmly anchored to the ER membrane markedly increased, whereas the corresponding [ 35 S]CYP3A species in the cytosol and that dislocated from the ER membrane into the Na 2 CO 3 wash significantly declined (Fig. 6A, bottom). After a 4-h treatment of these cells with DDEP, CYP3A inactivation once again resulted in protein ubiquitination, as documented by the HMM [ 35 S]CYP3A species, but these ubiquitinated [ 35 S]CYP3A species remained firmly bound to the ER. Furthermore, unlike the findings in control cells with their full p97 complement, following p97 knockdown, almost no parent and/or HMM ubiquitinated [ 35 S]CYP3A species were dislocated and escaped into the cytosol (Fig. 6A, bottom). The relative quantitative differences in [ 35 S]CYP3A trafficking as parent and ubiquitinated species from the ER into the cytosol with and without p97 knockdown were verified by our PhosphorImager/ImageQuant analyses (Fig. 6B). Collectively, these findings attest to p97 indeed playing an obligatory role in the ER extraction of the monotopic CYP3A protein. The corresponding ubiquitination profiles of hepatic protein in microsomes, cytosol, and Na 2 CO 3 wash reveal a similar abrogation of ubiquitinated hepatic protein extraction into the cytosol with a corresponding accumulation of ubiquitinated hepatic proteins in the ER, thereby confirming the key role of p97 in the ERAD of other hepatic ER proteins (supplemental Fig. S2).

DISCUSSION
Collectively, our data suggest that despite the topological accessibility of ER-anchored CYPs 3A to the cytosolic 26 S proteasome, a functional p97 is essential for the extraction of both the native and DDEP-inactivated CYPs 3A from the ER membrane into the cytosol. This extraction entails the dislocation of the intact CYP3A proteins, including their N-terminal ER membrane signal anchor, into the cytosol. However, not all ERAD substrates require p97 for their extraction/retrotranslocation/delivery to the cytosol for their cellular turnover because some are efficiently extracted by the functionally similar AAA ATPases of the PA700 (19 S) proteasomal cap (75)(76)(77)(78)(79)(80)(81)(82). Our findings (Fig. 5B) reveal that this is clearly not the case with CYPs 3A, which require p97 in a nearly obligatory manner. Upon p97 knockdown and inhibition of the 20 S proteasomal proteases by MG262, parent and/or ubiquitinated CYP3A would be expected to accumulate in the cytosol FIGURE 6. Relative intracellular localization of parent and ubiquitinated HMM CYP3A species after p97 knockdown in cultured hepatocytes. Rat hepatocyte cultures were infected with control shRNA or p97 shRNA 1ϩ2 (Ϫp97) for 7 days. On the 8th day, they were subjected to 35 S-pulse-chase analyses. Two h after cold chase, some cells were treated with or without DDEP for 4 h as described in the legend to Fig. 4. Cells were harvested, and homogenates were subfractionated into cytosol and microsomes. CYP3A immunoprecipitates (45 l) from cytosol (Cytosol), Na 2 CO 3 -washed microsomes (Microsomes), and wash corresponding to the Na 2 CO 3 -solubilized material (Wash) were obtained as described under "Experimental Procedures" and subjected to SDS-PAGE analyses. The gels were dried and then exposed to PhosphorImager screens and visualized using a Typhoon scanner. A, a typical SDS-PAGE gel is shown as a representative of corresponding CYP3A immunoprecipitates from pooled hepatocyte cultures. The color wheel intensity code is as follows: white Ͼ magenta Ͼ red Ͼ orange Ͼ yellow Ͼ green Ͼ light blue Ͼ dark blue Ͼ black. B, the corresponding relative
p97 is an abundant cytosolic AAA ATPase chaperone that works in concert with its cochaperone adapters Npl4 and Ufd1 as a heterotrimeric complex in the retrotranslocation/ extraction of integral/luminal ER proteins (21,39,40,(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56). This p97 complex-mediated ERAD function is essentially energy-driven by ATP hydrolysis (45,49). Although we have not directly addressed the specific roles of its Npl4 and Ufd1 adapters in the present study, our previous findings have revealed that, along with p97, both of these adapters were also critically required for the ERAD of heterologously expressed CYP3A4 in yeast (19). We therefore infer by analogy to yeast that in rat hepatocytes, an intact functional p97-Npl4-Ufd1 complex is similarly involved in the delivery of ER-anchored CYP3A to the cytosolic 26 S proteasome for its subsequent degradation and that this process too is also critically dependent on ATP-derived energy. Taken together with our previously published findings (11)(12)(13)(14)(15)(16)(17), the present findings underscore that both the native and DDEP-inactivated CYP3A species are genuine ERAD-C substrates, subscribing to all of the requisite criteria of an ERAD-C process (29 -31).
The findings above also reveal that CYP3A ubiquitination occurs to a substantial degree in the ER while the P450 protein is still tethered to the ER membrane. CYP3A ubiquitination thus precedes and to a large extent can be independent of CYP3A extraction into the cytosol. In vitro CYP3A4 ubiquitination analyses with functionally reconstituted recombinant mammalian enzymes have identified two effective E2-E3 systems: (i) UBC7 (an ERAD-associated Ub-conjugating enzyme) and gp78 (a polytopic ER-integral E3 Ub ligase) and (ii) UbcH5a (a Ub-conjugating enzyme) and CHIP (a cytosolic E3 Ub ligase) that acts in concert with Hsp70/Hsp40 chaperones (33)(34)(35). Our in vivo RNAi analyses targeted against either of these Ub ligases in cultured rat hepatocytes not only have confirmed this dual involvement but have also revealed that, physiologically, a substantial cellular fraction of native (non-DDEP-inactivated) CYP3A that is targeted by both of these E2-E3 systems is functionally active (35). These findings are fully congruent with our present findings of p97 RNAi, wherein a substantial fraction of hepatic CYP3A content is also found to be functionally active upon p97 knockdown (Fig.  3D). This is consistent with the relative increase in the content of the unmodified parent [ 35 S]CYP3A (55-kDa species) observed upon p97 knockdown in microsomes (Fig. 6A).
Parallel assessment of the relative immunodetectable CYP3A content in cultured hepatocytes following RNAi targeted against hepatic p97, gp78, or CHIP, through quantification of the relative immunofluorescence intensity of a 20-m 2 cytoplasmic area by confocal immunofluorescence microscopic analyses revealed values (mean Ϯ S.D.) of 116 Ϯ 6.3, 84.5 Ϯ 4.3, and 69.4 Ϯ 3.6 versus 47.2 Ϯ 4.8 for corresponding control shRNA-infected hepatocytes (Fig. 3C) (35). These findings suggest that CYP3A accumulation in hepatocytes was considerably higher after p97 knockdown than after knockdown of either gp78 or CHIP Ub ligase. They are consistent with the finding that p97 knockdown led to the ER accumulation of both the parent and ubiquitinated CYP3A species (Fig.  4). However, despite an otherwise effective ER protein ubiquitination process and no significant impairment of either gp78 or CHIP Ub ligase (Fig. 2C), this fraction of functionally active CYP3A protein accumulating after p97 knockdown was apparently comparable to that stabilized by the RNAi knockdown of either gp78 or CHIP (35). This suggests that either (i) the p97 complex can directly target both the parent non-ubiquitinated and ubiquitinated CYP3A species, consistent with its known targeting of both the ubiquitinated and the nonubiquitinated domains of ERAD substrates (45,47), or (ii) functionally active parent CYP3A accumulates upon p97 knockdown because it is possibly spared Ub modification due to exhaustion of available free Ub stores as follows: p97 knockdown impairs the extraction/retrotranslocation into the cytosol of all ubiquitinated ER proteins (including CYPs 3A) that are p97-dependent (supplemental Fig. S2). This ER accumulation of ubiquitinated proteins coupled with their impaired 26 S proteasomal processing would concurrently reduce recycling of poly-Ub chains required for fresh cycles of protein ubiquitination. In turn, this would spare a fraction of ER proteins, including CYPs 3A, from being normally de novo ubiquitinated, thereby retaining this population in the native functionally active form. Although in the case of other ERintegral proteins, such as the inositol 1,4,5-triphosphate receptors and Ste6p*, evidence has been provided that p97 targets only the ubiquitinated species (26,50) and thus acts postubiquitination but before proteasomal degradation of these ERAD substrates, this remains to be conclusively established in the case of p97 targeting of CYPs 3A. Our findings (Fig. 6A) revealing the dislocation of the unmodified parent [ 35 S]CYP3A (55-kDa) species into the cytosol and to the external surface of the ER membrane (Na 2 CO 3 -wash) in the presence of a fully functional p97 complement suggest that protein ubiquitination may not be absolutely essential for the extraction of all CYP3A molecules. 4 We also find it noteworthy that, upon p97 knockdown, a substantial fraction of DDEP-inactivated CYP3A species not only is similarly ubiquitinated while firmly ER-anchored and accumulates in this form, but also its ubiquitination is considerably enhanced and extends to an even higher molecular mass range upon MG262-mediated proteasomal inhibition (Fig. 5B). Such enhanced CYP3A ubiquitination may result from the delay in CYP3A dislocation, thereby providing addi- 4 Although our data are consistent with both ubiquitinated and the parent species being extracted out of the ER (microsomes) by p97, our recent findings (35) reveal that such extraction of the parent species requires CHIP E3 Ub ligase as well. Knockdown of CHIP but not gp78, even in the presence of a fully functional p97, virtually abolishes such parent CYP3A extraction into the cytosol (35). The inclusion of N-ethylmaleimide (5 mM), an inhibitor of deubiquitinating enzymes, in our harvesting/lysis buffer should exclude the possibility that this cytosolic "parent" CYP3A species stems from CYP3A deubiquitination following lysis of the cells. FEBRUARY 4, 2011 • VOLUME 286 • NUMBER 5 tional time for the elaboration of longer Ub chains and/or the appendage of Ub chains on additional CYP3A residues. Together, these findings are entirely consistent not only with a major role for p97 in the dislocation of the parent as well as the ubiquitinated CYP3A species into the cytosol, but also with CYP3A ubiquitination by the ERAD-associated Ub ligases gp78 and CHIP occurring while the proteins are still tethered to the ER membrane (35). The functional involvement of both gp78 and p97 in CYP3A UPD/ERAD is fully consistent with the intimate structural-functional association of these two proteins that effectively couples the ubiquitination of numerous integral and luminal ER proteins to their ER retrotranslocation/dislocation during their ERAD (49,58,(83)(84)(85)(86). Accordingly, the ER polytopic gp78 Ub ligase protein is proposed to contain a p97-docking domain, the VIM, in its cytosolic C terminus that serves to recruit p97 to its ERbound ubiquitinated substrate 5 (58,(83)(84)(85). Whether a similar direct functional association exists between p97 and CHIP remains to be established. Some evidence exists in support of such a functional p97-CHIP association (87,88).

p97-mediated CYP3A ER Extraction into the Cytosol
The ER also contains the monotopic VCP-interacting membrane protein (VIMP), which is proposed to recruit p97 to the ER membrane and, together with p97, to function in a hetero-oligomeric complex with Derlin-1, another polytopic homo-oligomeric ER membrane protein (46,49). Together, this p97-VIMP-Derlin-1 complex is reportedly involved in the retrotranslocation of misfolded and luminal ER proteins across the ER membrane into the cytosol for delivery to the 26 S proteasome (49). Whether Derlin-1 and/or VIMP are also involved in CYP3A ERAD remains to be determined. The direct interaction of p97 with gp78 as well as other ERADassociated Ub ligases (i.e. Hrd1 and CHIP) purportedly enables p97 recruitment of these E3s to the ER substrate in question within the Derlin-1 translocation channel in the ER membrane. However, our findings indicate that this is not absolutely essential for CYP3A ERAD because CYPs 3A are quite efficiently ubiquitinated in the ER following such an extensive p97 knockdown (Figs. 4B, 5B, and 6A).
To our knowledge, this is perhaps the most extensive (Ͼ90%) cellular knockdown of this abundant cytosolic p97 protein reported in the literature. Given the vast array of cellular functions dependent on p97 (55-61), we find it both surprising and remarkable that our confocal immunofluorescence microscopic analyses attested to relatively healthy viable hepatocytes with no disruption of their cellular morphology or any detectable cell necrosis after 7 days of such an extensive p97 knockdown (Fig. 3C). No evidence of extensive cytoplasmic vacuolization, ER stress and expansion, impaired cell function, cell death, and/or apoptosis was found (Fig. 3C), as reported in neuronal PC12 cells expressing a dominant negative p97 (89) or HeLa cells transiently transfected with synthetic siRNAs that knocked down p97 by ϳ80% (60,61). For comparison, corresponding analyses of similarly cultured hepatocytes treated with the proteasomal inhibitor MG-132 (300 M), a concentration known to induce ER stress as determined by the activation and/or induction of hepatic ER stress markers PERK and GCN2 eIF2␣ kinases as well as the elevation of hepatic Grp78/Bip protein levels (66), are included (supplemental Fig. S3). qRT-PCR analyses of total RNA from control and p97-knocked down hepatocytes revealed no significant increases in hepatic Grp78 and Grp94 mRNA content (Fig. 2C), sensitive indices of a full-blown unfolded protein response/ER stress response (90). Furthermore, none of the prototypic morphologic hallmarks of apoptosis, such as chromatin condensation, collapse, or budding and/or significant nuclear shrinkage or fragmentation were detected at this time (Fig. 3C), in contrast to those observed in cultured hepatocytes treated with high MG132 concentrations (300 M; supplemental Fig. S3) (66). Indeed, not only were the nuclei morphologically similar to control nuclei, but also the nuclear size of the p97-knocked down hepatocytes was comparable to that of the corresponding control shRNA-treated hepatocytes (Fig.  3C). In addition, cellular processes, such as protein ubiquitination (including that of CYPs 3A) (supplemental Fig. S2), CYP3A synthesis, 6 and functional activity (Fig. 3D), and proteasomal function (Fig. 5A) were unaffected following this extensive 7-day p97 knockdown. Thus, it appears that such an extensive knockdown, albeit impairing hepatic ERAD/UPD of CYP3A proteins and resulting in the accumulation of ubiquitinated proteins, is relatively well tolerated by the hepatocyte. Accordingly, just ϳ10% of functional hepatic p97 protein content apparently is sufficient and compatible with cellular survival and health of sandwich-cultured primary rat hepatocytes at least for a day or so after the 7-day p97 knockdown. Thus, primary hepatocytes appear to be strikingly more tolerant of extensive p97 knockdown than cell lines, and whether this tolerance extends to other primary cells remains to be determined. Alternatively, the lentiviral approach of shRNA delivery that entails integration of the silencing sequence into the hepatocyte chromosome, albeit slower in producing the desired knockdown, may enable the cells to adjust more gradually to the changes in their intracellular milieu and thus to better withstand the cellular stress of p97 knockdown. p97 knockdown of this extent clearly impairs CYP3A ERAD, resulting in the stabilization of a functionally active CYP3A, which in the human liver could lead to clinically relevant drug-drug interactions. However, it remains to be determined whether a less severe p97 impairment due to genetic 5 It is noteworthy, nevertheless, that mutation of gp78 residue 611 in this domain that reportedly abolishes gp78 binding to p97 (58) failed to abolish the degradation of gp78 itself and of its heterologous substrate CD3-␦ (41). 6 This is deduced from our qRT-PCR analyses indicating that p97 knockdown failed to affect hepatic CYP3A mRNA levels (Fig. 2B) and our observation that the [ 35 S]radioactivity (cpm/mg of lysate protein/h) incorporated into CYP3A immunoprecipitates (Fig. 6) at time 0 h following pulse-chase was equivalent: 77,434 Ϯ 2560 (control) versus 77,000 Ϯ 2554 (Ϫp97).

p97-mediated CYP3A ER Extraction into the Cytosol
polymorphisms or faulty expression of allelic variants is at all encountered clinically and would influence P450-dependent function and consequent drug-drug interactions. The expression of a p97 mutant with dominant negative mutations in the catalytic domains of both of its ATPase subunits is indeed capable of impairing UPD (45,89). Thus, impaired CYP3A ERAD may be of clinical relevance in individuals with p97 proteins with functional defects in their ATPase domains and/or gp78/CHIP-interacting domains if such defects were not embryonically lethal. In summary, our findings reveal that p97 plays a critical physiological role in CYP3A ERAD by extracting the CYPs 3A from the ER membrane into the cytosol and subsequently delivering them to the 26 S proteasome for destruction. p97 knockdown via RNAi abrogates this process and results in the accumulation of functionally active CYP3A species as well as extensively ubiquitinated species tethered to the ER membrane.


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Identification and Characterization of Endogenous Galectins Expressed in Madin Darby Canine Kidney Cells*

Madin Darby canine kidney (MDCK) cells are a well characterized epithelial cell line used to study mechanisms of polarized delivery. As glycans on apically expressed proteins have been identified as targeting signals, and crosslinking by the abundant galectin-3 has been implicated in the mechanism of glycan-dependent sorting, we wanted to identify other members of the galectin (Gal) family expressed in MDCK cells. By analyzing intron-exon boundaries, we identified canine genes that were highly homologous to mammalian Gal-1, 2, 3, 4, 7, 8, 9, and 12, and galectin-related HSPC159 and GRIFIN. Transcripts for Gal-2 and -12 were not detected in MDCK cells, but we found transcript levels for Gal-3 > Gal-9 > Gal-8 > Gal-1 ⋙ Gal-4 > Gal-7. Canine Gal-1, -2, -3, -4, -7, -8, -9, and -12 were cloned and expressed in Escherichia coli as GST fusion proteins to characterize binding specificities on arrays of synthetic glycans on glass slides from Core H of the NIH Consortium for Functional Glycomics. Individual expression of the N-terminal (GST-Gal-9N) and C-terminal (GST-Gal-9C) carbohydrate recognition domains greatly improved protein yield and the ability to characterize Gal-9 binding on the array. Canine galectins differentially bound sulfated disaccharides as well as human blood groups A, B, and H on both N-glycans and linear glycan structures on the array. Analysis of GST-Gal-1, -3, -4, -7, -8, -9N, and -9C binding to immunopurified human MUC1 expressed in MDCK cells revealed a preference for binding GST-Gal-3 and -9, which interestingly reflects the two most abundant galectins expressed in MDCK cells.

The functions of glycans on glycoproteins are quite varied, with immature N-linked glycans playing a key role in protein folding and quality control within the endoplasmic reticulum, and mature terminally processed N-and O-linked glycans playing key roles in glycoprotein interactions as diverse as subcellular targeting, signal transduction, cell-matrix interactions, cell-cell interactions, and cell-microbe interactions (1). We are specifically interested in the role of glycans in apical targeting in polarized epithelial cells as N-glycans on secreted model proteins, and both N-glycans and O-glycans on transmembrane model proteins, have been identified as apical targeting signals (for review, see Refs. 2,3).
Recent studies on the mechanism of apical targeting in the model system of polarized Madin Darby canine kidney (MDCK) 3 epithelial cells has revealed that proteins with unique types of apical sorting signals are differentially sorted into subpopulations of vesicles budding from the trans-Golgi network (TGN) (4 -6). There is also evidence that some apical proteins traffic through unique endocytic compartments within the biosynthetic pathway such that proteins like influenza hemagglutinin and GPI-anchored proteins that exhibit a lipid raft-dependent targeting signal, transit the apical early endosome (AEE), while proteins like endolyn and p75 neurotrophin receptor that exhibit an N-glycan-and O-glycan-dependent targeting signal, respectively, transit the apical recycling endosomes (ARE) (7,8). MUC1 is a transmembrane glycoprotein with a mucin-like ectodomain that is localized on the apical surface of epithelial cells; it provides protection from pathogens and likely plays a key role in epithelial development, repair and tumorigenesis (for review, see Ref. 9). Although the targeting signal for MUC1 apical delivery in polarized epithelial cells has not been defined, studies with MUC1 chimeras in MDCK cells did reveal that the signal is within the mucin-like ectodomain of MUC1 (10). We also observed that apical delivery of MUC1 in polarized MDCK cells was blocked by ablation of the ARE, but not the AEE, consistent with a glycan-dependent apical targeting signal for MUC1 (11). Interestingly, Delacour et al. (12,13) reported that vesicles budding from the TGN carrying lipid-raft-independent cargo contain galectin-3, and that galectin-3 crosslinking of the glycoprotein cargo is required for apical sorting. As galectin-3 (Gal-3) constitutes only one member of the family of galectins and MUC1 interactions with Gal-3, and correlative expression with Gal-1 and -4 have been reported (14 -17), we were interested in the identification, cloning, and characterization of all canine galectins expressed in MDCK cells.
The family of galectins is characterized by (i) their affinity for binding glycoconjugates containing ␤-galactose, (ii) their homologous sequence and structural elements, and (iii) their conserved exon/intron junctions (18 -21). Galectins were previously named S-type lectins as many in this group are stabi-lized by the presence of thiols, and oxidation of the invariant Cys correlates with inactivation. There are more than a dozen galectins described in the literature and they are more or less numbered based on the order of their discovery although they fall into three subgroups. Prototypical galectins like Gal-1, with a single carbohydrate recognition domain (CRD) of about 130 residues, usually form dimers. Other members of this subgroup include Gal-2, -7, -10, -13, -14, -15, and galectin-related proteins PP13, PPL13, HSPC157, and GRIFIN (the latter four lack essential glycan-interacting residues and thereby the ability to bind glycoconjugates). Gal-3 comprises the only member of the chimeric subgroup with one CRD and a unique N-terminal domain involved in formation of multimers. Members of the third subgroup termed tandem-repeat, have two non-identical CRD joined by a linker domain, and include Gal-4, -6, -8, -9, and -12. Interestingly, a phylogenetic analysis of gene locations, exon-intron structures and sequences of chordate galectins revealed that Gal-1 is not the prototypical galectin (22). Instead, duplication of a mono-CRD galectin gene early in chordate evolution gave rise to a bi-CRD galectin gene, and independent evolution of the Nterminal CRD and C-terminal CRD gave rise to two distinct mono-CRD types including Gal-7, -10, -14, PP13, and PPL13 in the F4-group, and Gal-1, -2, -3, -5, HSPC159, and GRIFIN in the F3-group, respectively (22). Although canine Gal-3 and Gal-8 were included in this latter analysis, there have been no additional studies of canine galectin genes despite the availability of the dog genome and the widespread interest in the well-characterized model of MDCK cells. Friedrichs et al. (23) did estimate the levels of transcripts for some of the galectins in MDCK cells using real-time polymerase chain reaction (PCR). We now report the identification of eight canine galectin genes (and two galectin-like genes), of which six galectins are expressed in MDCK cells. We have cloned the canine galectin cDNAs and expressed them as GST-tagged proteins in order to assess their binding specificities on synthetic glycan arrays and their differential interactions with MUC1 synthesized in MDCK cells.

MATERIALS AND METHODS
Amplification of Canine Galectin RNA by RT-PCR-RNA from dog jejunum and heart (breed, Beagle) was purchased from Zyagen (San Diego, CA). MDCK type II cells (breed, Cocker Spaniel) were obtained from Gerard Apodaca (University of Pittsburgh), and RNA was isolated using Ambion RNAqueous 4PCR Kit (Austin, TX) as described by the manufacturer. First strand cDNA was synthesized from 1 g RNA using RNA Superscript II reverse transcriptase and amplified using TaqDNA polymerase (Invitrogen, Carlsbad, CA). cDNAs were amplified using internal primers listed in supplemental Table S1 by heating at 95°C for 30 s, annealing primers at 56°C for 30 s and polymerization at 72°C for 30 s. Amplified DNA (ϳ200 bp) for Gal-1, -3, -8, and -9 was visible on ethidium bromide-stained agarose gels after 28 cycles. Amplified cDNAs for Gal-2, -4, -7, and -12 were prepared with nested primers using the amplified product obtained with external primers after 28 cycles as substrate for a second amplification with the internal primers for 28 cycles.
Cloning of Canine Galectin cDNAs by RT-PCR-First strand cDNA was synthesized from heart, jejunum and MDCK RNA as already described and amplified using external primers listed in supplemental Table S1 by heating at 95°C for 30 s,  annealing primers at 56°C for 30 s and polymerization at  72°C for 60 s. Amplified cDNAs for Gal-1, -3, -4, -7, and -8  prepared from MDCK RNA, amplified cDNAs for Gal-2 and  -9 from dog jejunum RNA, and Gal-12 from dog heart RNA were cloned into pCR2.1 TOPO vector from Invitrogen. Amplified cDNAs for Gal -7, -9, and -12 were prepared with nested primers using the amplified product obtained with UTR primers after 28 cycles as substrate for a second amplification with the external primers for 28 cycles. Multiple plasmid preps for each cloned cDNA were subjected to nucleotide sequencing using M13rev and T7 primers (GenBank TM accession numbers HQ637384-HQ637391 for eight galectins).
Expression of GST-galectins in Bacteria-Gal-1, -2, -3, -4, -7, -8, -9, and -12 cDNAs were subcloned into pGEX-6P-1 for expression in bacteria as N-terminal GST fusion proteins as previously described (24). As GST-Gal-9 repeatedly aggregated in the bacteria, we subcloned the N-terminal and Cterminal CRDs separately as GST-Gal-9N (residues 1-148) and GST-Gal-9C (residues 225-355), respectively, for further study. GST-Gal-12 also aggregated and was not further studied. Briefly, 2 liters of liquid culture were inoculated with E. coli strain BL21 transformed with pGEX-6P-1(GST-galectin). Isopropyl ␤-D-1-thiogalactopyranoside was added at mid-phase (OD ϳ0.8), and bacteria were grown with shaking overnight at room temperature. The supernatant from the lysed bacteria was incubated at 4°C overnight with 0.75 ml of a 50% slurry of glutathione-conjugated Sepharose (GE Healthcare, Piscataway, NJ) in a 15-ml conical tube with end-overend mixing. GST-galectins were released from the washed beads with 1 ml of buffer (0.05 M Tris-HCl, pH 8, 150 mM NaCl) containing 14 mM ␤-mercaptoethanol and 15 mM glutathione. After centrifugation, the supernatant was incubated with 1 ml of a 50% slurry of lactose-conjugated Sepharose (Sigma) in a 15-ml conical tube at 4°C for 30 min with endover-end mixing. The slurry was poured into a 1-cm diameter column containing a sintered glass filter and the packed column bed was overlaid with 1 ml of a 50% slurry of Sepharose 6B (Sigma). The column bed was washed with 5 ml of buffer containing 14 mM ␤-mercaptoethanol (␤ME). 2 ml of buffer containing ␤ME and 0.1 M ␣-lactose (Sigma) were added to the column, 0.5 ml collected, and the column capped for 30 min at 4°C. Fractions of 0.5 ml were collected, and the absorbance at 280 nm was determined with a spectrophotometer. The peak fractions were pooled and lactose was removed on a PD-10 column as described by the manufacturer (GE Healthcare). Final protein concentrations were calculated using individual extinction coefficients determined from the amino acid content of each GST-Gal.
Glycan Array Screening of Canine GST-galectins-Freshly prepared GST-galectins in buffer containing ␤ME were shipped overnight on ice to Core H of the Consortium for Functional Glycomics for analysis on glycan microarrays. GST-galectins were diluted to 0.1-100 g/ml in standard binding buffer (Tris-buffered saline with MgCl 2 and CaCl 2 , 1% BSA and 0.05% Tween 20). Seventy microliters were applied to the printed surface of the array before applying the coverslip and incubation at room temperature in a humidified chamber away from light for 1 h. The coverslip was removed and the slide was rinsed four times in standard TSM washing buffer containing 0.05% Tween and four times in TSM buffer without Tween. Seventy microliters of Alexa 488 conjugated anti-GST antibody (Invitrogen) was diluted in TSM buffer and applied for one hour in a humidified chamber away from light as described. Washes were performed as above, followed by four washes in distilled water before fluorescence of the spots was measured.
MUC1 Pull-down with Recombinant GST-galectins-Polarized MDCK type II cells growing on Costar permeable supports (Corning, NY) were cultured as previously described (7,11,25). Where indicated, MUC1 expression was achieved by infection with recombinant adenovirus (7,11). A clonal MDCK cell line expressing MUC1 was previously described (7). Polarized MDCK cells expressing MUC1 were pulse labeled for 30 min with [ 35 S]Met/Cys and chased for 90 min, and immunoprecipitates recovered with mouse monoclonal antibody B27.29 and protein G-conjugated Sepharose as previously described (7,11). Proteins were eluted from protein G-conjugated Sepharose by incubation at 90°C for 2 min in buffer A (10 mM HEPES, pH 7.4, 150 mM NaCl) containing 1% SDS. The eluted material was diluted 10-fold with buffer A containing 1% Triton X-100 (and adjusted to 14 mM ␤ME). Identical samples from six 12 mm filters of polarized MDCK cells were combined and equal aliquots were incubated with either GST-Gal-1, -3, -4, -7, -8, -9N, or -9C prebound to glutathioneconjugated Sepharose (or no GST-Gal as a control), overnight on a rotating wheel at 4°C. A fresh aliquot of each GST-galectin was prepared for every experiment by affinity purification (starting material ϳ1 g) on lactose-conjugated Sepharose (25 l of a 50% slurry) and removal of lactose after subsequent binding to glutathione-conjugated Sepharose (25 l of a 50% slurry) as described above. The following day, the beads were washed with buffer A and proteins eluted by successive incubations for 15 min each on a rotating wheel at 4°C with buffer A containing (i) 0.1 M sucrose (as control), (ii) 0.1 M lactose (for specific binding) and (iii) 15 mM glutathione (to recover GST-galectins). The eluents (10 l) were recovered with a Hamilton syringe after centrifugation (10,000 ϫ g for 30 s) and mixed with 10 l of Bio-Rad sample buffer for SDS-PAGE on a precast 4 -15% gradient gel (Bio-Rad) and transfer to nitrocellulose (Millipore). Radiolabeled proteins were detected with a Bio-Rad Imager using a TR phosphorimager screen before cutting the nitrocellulose at the 100 kDa marker for immunoblotting of the large or small subunit with mouse monoclonal antibody B27.29 or Armenia hamster monoclonal antibody CT-2, respectively. Bands were detected using Perkin Elmer Western Lightning TM Plus-ECL with a Bio-Rad Versadoc or Kodak BioMax MR film, and GST-galectins were detected by Coomassie staining (Bio-Rad Bio-safe Coomassie). The levels of radiolabeled proteins, immunoblotted subunits or Coomassie-stained GST-galectins were determined with Bio-Rad Quantity One software. The level of galectin binding of radiolabeled or immunoblotted subunits was nor-malized to the level of galectin recovered for the same sample based on Coomassie staining (divided by the molecular weight of the galectin as reported in Table 1). In each experiment, maximal binding to a single galectin was set as 1 and binding to other galectins was calculated as a fraction of maximal binding. Data were combined by averaging the values for four experiments (presented as mean and S.E.).

Identification of Canine Galectin Genes-
The genes for canine Gal-1, -2, -3, -4, -5, -8, -9, and -12 as well as galectinrelated GRIFIN, were identified by a query of the dog genome for the term "galectin" (breed, Boxer). Identical references to canine genes were found with a query of the NCBI Nucleotide Database and searches using Nucleotide BLAST and TBLASTN. A pseudogene for Gal-1 containing a polyadenylation motif and no introns was also reported on the X chromosome and was not analyzed further. The predicted coding sequence of the Gal-4 gene included three CRDs but upon closer study, actually included the genes for both Gal-4 and Gal-7. A study of the gene attributed to Gal-5 revealed that it was actually homologous to mammalian galectin-related HSPC159. No canine genes homologous to rat Gal-5, mouse Gal-6, human Gal-10, human Gal-13, ovine Gal-14, ovine Gal-15, human PP13, or human PPL13 were identified. In general, genes for canine Gal-1, -2, -3, -4, -7, -8, -9, -12, HSPC159, and GRIFIN were highly homologous to the corresponding mammalian galectin genes with similar exon/intron structures and amino acid sequence (see Table 1, Fig. 1, and supplemental Figs. S1-S8). Canine GRIFIN and HSPC159 lack two and five essential residues, respectively, in the CRDs for sugar binding so they were not further investigated in this study. Primers for amplification of galectins from canine RNA by RT-PCR are described in supplemental Table S1. External primers overlapped the Met start codon and the stop codon in each case. UTR primers were designed within 5Ј and 3Ј UTR as needed. Internal primers were designed from exon sequences to produce a ϳ200-bp product.
Identification of Endogenous Galectins Expressed in MDCK Cells-RNA was prepared from MDCK cells and amplified by RT-PCR as described in Materials and Methods to yield ϳ200-bp products. RNA from a commercial source of dog jejunum was used as a positive control. RNA for Gal-1, -3, -8, and -9 was most abundant in both MDCK and jejunum as the ϳ200 bp products from RT-PCR were visible on agarose gels with ethidium bromide staining using only internal primers ( Fig. 2A). Amplification of RNA from Gal-2, -4, -7, and -12 was apparent only with nested primers; the ϳ200 bp product of amplification was produced only when the product from RT-PCR with external primers was subsequently used as template for PCR with internal primers. The data indicated that all the canine galectins could be amplified with the selected primers using jejunum RNA as a positive control, but only Gal-1, -3, -4, -7, -8, and -9 were amplified from MDCK cell RNA. The product of ␤-actin amplification by RT-PCR from jejunum and MDCK cell RNAs was similar.
The cloned canine Gal-1 cDNA had the same nucleotide sequence as predicted by a query of the dog genome. The division of the coding sequence between the four exons of the canine gene was identical to the human Gal-1 gene and there was 82% identity in amino acid sequence.
The cloned canine Gal-2 cDNA had the same nucleotide sequence as predicted by a query of the dog genome (breed, Boxer) except that we found Val-124 is Leu-124 in our cloned cDNAs (breed, Beagle). Interestingly, the division of the coding sequence between exons 3 and 4 (four exons total) was different than reported for the human Gal-2 gene but the amino acids are identical (77% identity between human and canine Gal-2).
The cloned canine Gal-3 cDNA had the same nucleotide sequence as predicted by a query of the dog genome including six 9-mer repeats in coding exon 2. There are a variable number of 9-mer repeats (Q-A/G-P-P-G-G/A/PT-Y-P-G) reported for cow (four), mouse (three), pig (three), human (two), rat (two), and rabbit (one) Gal-3 (see asterisk in Fig.  1D). Otherwise, the division of the coding sequence between the five exons was identical to the human Gal-3 gene (with 73% identity of amino acids). The exon containing the 5Ј-UTR has not been identified for the canine Gal-3.
The cloned canine Gal-4 cDNA sequence was very different from that predicted by the canine genome data base where it is fused with a sense and antisense copy of canine Gal-7. The division of the coding sequence between the nine exons was identical to the human Gal-4 gene except that the canine Gal-4 is missing one exon of 27 bp (9 amino acids) between exons 4 and 5 (with 77% identity of amino acids) (see asterisk in Fig. 1E).
The cloned canine Gal-7 cDNA was very different from that predicted by the canine genome data base where its sequence is fused with canine Gal-4 and predicted to have an additional exon between exons 3 and 4. In our cloned sequence, the division of the coding sequence in exons 2, 3, and 4 was identical to the human Gal-7 gene with 76% identity of amino acids. There are a variable number of residues within the first coding exon of mouse (two), dog (five), cow (five), horse (six), and human (six).
The cloned canine Gal-8 cDNA had the same nucleotide sequence predicted by the canine genome database, except that it lacks the seventh coding exon (123 bp) within the linker region and thereby corresponds to the short isoform of Gal-8 (see asterisk in Fig. 1F). The division of the coding sequence between exons 2-9 of the canine gene was identical to the human Gal-8 gene, while the coding sequence in the first exon is one residue shorter. There is 86% identity between the human and canine Gal-8 short isoforms.
The cloned canine Gal-9 cDNA has the same nucleotide sequence as that predicted by the canine genome database, except that it includes exon 5 (96 bp) and thereby represents the long isoform (see asterisk in Fig. 1G). Our clone also had Thr-216 instead of Met-216, and Met-281 instead of Thr-281. The division of the coding sequence between the eleven exons of the canine gene was identical to the human Gal-9 gene and there was 74% identity of amino acids.
The cloned canine Gal-12 cDNA has the same nucleotide sequence as that predicted by the canine genome database, except that it lacks an exon (27 bp) between exons 5 and 6 and thereby represents the short isoform (see asterisk in Fig.  1H). Our clone also had Leu-300 instead of Val-300. The division of the coding sequence between the eight exons of the canine gene was identical to the human Gal-12 gene with 85% identity of amino acids. The exon containing the 5Ј-UTR has not been identified for the canine Gal-12.
Characterization of Glycan Binding for Canine Galectins-The cDNAs for canine Gal-1, -2, -3, -4, -7, -8, -9, and -12 were subcloned into the bacterial vector pGEX-6P-1 for expression in E. coli as N-terminal GST fusion proteins as previously de- scribed (24). The fusion proteins were affinity purified on glutathione-conjugated Sepharose and then lactose-conjugated Sepharose to obtain epitope-tagged galectins for characterization on the NIH Consortium Core H glycan array. GST-Gal-2, -9, and -12 consistently gave low yields, most likely due to aggregation in bacterial inclusion bodies (see Fig. 2B for a Coomassie-stained gel of all the GST-Gal). As Gal-9 is expressed in MDCK cells, which is the focus of this study, we attempted to improve the yield by sub-cloning the two carbohydrate recognition domains separately. Expression of GST-Gal-9N (residues 1-148) and GST-Gal-9C (residues 225-355) in bacteria gave yields similar to those obtained for other affinity purified GST-galectins (0.2-0.5 mg total from a 2-liter culture) (Fig. 2B). Binding of Gal-1, -2, -3, -4, -7, -8, -9N, and -9C to glycan arrays on glass slides was carried out at multiple concentrations and analyzed with a fluorescently tagged anti-GST antibody. Greater specificity was consistently revealed at the lower concentrations and the preferred binding was determined by comparing the ranked order at multiple concentrations as previously described (26). Briefly, each GST-Gal was analyzed at multiple concentrations. At each concentration the Relative Fluorescence Units (RFU) corresponding to the glycan with the highest binding signal was set at 100 and the relative binding of the other glycans was calculated to provide a rank order of binding. The average of the three rankings for each glycan at three concentrations of GST-Gal was determined and the bound glycans were ordered from highest to lowest ranking as shown in supplemental Tables S2-S9. A representative profile at a single concentration for each GST-Gal is presented in Fig. 3. Structures of bound glycans are indicated and the average rank (in red numbering with 1 as the best binding) is provided in each case for comparison to the   Table S1 for primer sequences. B, cDNAs for Gal-1, -2, -3, -4, -7, -8, -9, and -12 were expressed in bacteria as GST fusion proteins and subjected to SDS-PAGE and Coomassie staining. The N-terminal and C-terminal carbohydrate recognition domains of Gal-9 were individually expressed as GST-Gal-9N and GST-Gal-9C, respectively. representative profile. A summary of the results are addressed below. Array data are also available online.
GST-Gal-2 was analyzed at two concentrations (5 and 50 g/ml) and showed notable preference for biantennary Nglycans with terminal Gal␣1,3 extended Lewis x , with or without core GlcNAc fucosylation (see supplemental Table S3 and Fig. 3B). Binding to other glycans was generally 100-fold lower.
GST-Gal-4 binding was determined at three concentrations (0.5, 5, and 50 g/ml) and preferentially bound linear glycans with terminal blood group A (3, 4, 6) or Gal␤1,3 extended structures (1) as shown in supplemental Table S5 and Fig. 3D. Blood group A on a biantennary N-glycan (5) had reduced binding when compared with blood group A on lactose (4) or PL (3). Blood group B on lactose (2) or biantennary N-glycan (8) was also bound.
GST-Gal-7 binding was determined at three concentrations (0.1, 1, and 10 g/ml) as shown in supplemental Table S6 and Fig. 3E and preferentially recognizes blood group H, but only on type 2 PL (1 and 2). Termination of type 2 PL chains with Neu5Ac␣2,6 (6 and 7) or blood group A (5 and 8), instead of fucose, decreased binding somewhat, whereas termination with Neu5Ac␣2,3 or blood group B was not tolerated.
GST-Gal-8 binding was determined at three concentrations (0.1, 1 and 10 g/ml) as shown in supplemental Table S7 and Fig. 3F and preferentially bound poly-NAc-lactosamine (PL) with various modifications, as well as biantennary Nglycans with blood group A (2) (weaker for blood group B, 7). Binding to PL terminating with Gal (6) was enhanced by addition of terminal Neu5Ac␣2,3Gal␤1,4 (1) but not GlcNAc alone (5), and was reduced substantially by addition of terminal Neu5Ac␣2,6Gal␤1,4. Binding of PL terminating in blood group A was enhanced by longer PL chains (compare 3 and 9).
GST-Gal-9N binding was determined at three concentrations (0.1, 1, and 5 g/ml) as shown in supplemental Table S8 and Fig. 3G and showed preferential binding to blood group A as well as 3-O-sulfated disaccharides. Bivalent blood group A found on biantennary N-glycans (1) was bound better than monovalent blood group A found on either lactose (8) or PL (4 and 7). Binding to blood group B on biantennary N-glycans (12) was 6-fold lower than the corresponding N-glycan with blood group A (1).
GST-Gal-9C binding was determined at three concentrations (0.1, 1, and 5 g/ml) as shown in supplemental Table S9 and Fig. 3H and showed preferential binding to primarily sulfated disaccharides (eight of top ten). Binding to blood group B on a biantennary N-glycan (6) and PL with terminal Neu5Ac␣2,3 (8) was also observed.
As canine HSPC159 was initially listed as Gal-5 in the dog genome data base, we also cloned the corresponding cDNA and expressed it as "GST-Gal-5." This GST-Gal-5 did bind and elute from the glutathione column but it did not subsequently bind to lactose conjugated to Sepharose during the purification step. 4 This lack of binding was consistent with the lack of five essential residues within its carbohydrate recognition domain.
Differential Binding of MUC1 Subunits to GST-galectins-To evaluate the potential interaction of canine galectins with MUC1 in MDCK cells, we assessed the ability of the recombinant canine galectins to precipitate immunopurified MUC1 expressed in MDCK cells. Polarized cultures of MDCK cells expressing MUC1 were metabolically labeled and MUC1 was immunoprecipitated from cell extracts as described under "Materials and Methods." The immunoprecipitates were recovered in buffer containing 1% SDS to dissociate the MUC1 large and small subunits and denature any bound endogenous galectins. MUC1 is synthesized as a single peptide but exhibits autocatalytic cleavage within the ectodomain yielding a small subunit containing the transmembrane domain and one N-linked glycan while the large subunit contains four N-linked glycans and the mucin-like tandem repeat domain with numerous sites for O-linked glycans. The samples were diluted into buffer containing 1% Triton X-100 and ␤-mercaptoethanol prior to mixing with the GST-galectins to stabilize recombinant galectin structure and activity. Fresh aliquots of each GST-galectin were affinity purified on lactose-conjugated beads immediately before binding to glutathione-conjugated beads and incubation with immunopurified MUC1. After overnight incubation at 4°C, the washed beads were incubated sequentially with buffer containing 0.1 M sucrose (control), 0.1 M lactose (specific-binding), and glutathione (GST-Gal recovery). MUC1 released by sucrose and lactose washes were analyzed by SDS-PAGE and transferred to nitrocellulose before analyzing the radiolabeled bands with a phosphorimager (Fig. 4A). The nitrocellulose was subsequently cut in half and immunoblotted for either the large (M r ϳ250,000) or small (M r ϳ25,000 or a dimer at M r ϳ50,000) subunit of MUC1 (Fig. 4B). Eluted GST-galectins were subjected to SDS-PAGE, the gel stained with Coomassie, and the bands quantified after scanning. One representative experiment is shown in Fig. 4 and the combined results from four independent experiments are shown in Fig. 5.
In general, we observed Ͼ10-fold greater recovery of MUC1 from GST-galectins bound to glutathione-conjugated beads when the beads were incubated with lactose (L) than with sucrose (S), consistent with specific binding of MUC1 to all of the canine galectins (Fig. 4, A and B, compare lanes S with L in each case). We also observed similar levels of each GST-galectin released from the beads with glutathione when analyzed by Coomassie staining (Fig. 4C). The combined results from four independent experiments reported in Fig. 5A indicated that the large subunit preferentially bound both GST-Gal-3 and GST-Gal-9N. No significant difference was observed for the results obtained by analysis of either radiolabeled or immunoblotted large subunit. Binding of the large subunit to GST-Gal-4, -7, and -9C was 2-3-fold less, while binding to GST-Gal-1 and GST-Gal-8 was 5-and 10-fold less, respectively. We consistently observed that the small subunit preferentially bound to GST-Gal-3 in every experiment. The combined results in Fig. 5B indicate that binding of the small subunit to GST-Gal-7, -9N, and -9C was 3-4-fold lower while binding to GST-Gal-1, -4, and 8 was 5-10-fold lower. We did observe a trend toward greater binding of the small subunit to GST-galectins other than GST-Gal-3 when analyzed by metabolic labeling than by immunoblotting.
Using RT-PCR we estimate that the levels of transcripts expressed in MDCK cells are Gal-3 Ͼ Gal-9 Ͼ Gal-8 Ͼ Gal-1 Gal 4 Ͼ Gal-7, as evidence of Gal-4 and Gal-7 transcripts was obtained only with nested primers. Using realtime PCR and primers based on the canine exon sequences, Friedrichs et al. found that Gal-3 transcripts were 100-times more abundant than transcripts for Gal-1, -8, and -9, and 100,000 times more abundant than transcripts for Gal-12 which is consistent with our findings (23). However, the same group mistakenly identified transcripts for Gal-6, but not Gal-4, using erroneous sequences for the PCR primers (exon sequences for canine Gal-4 were used for the sense primer to amplify Gal-4 and the antisense primer for Gal-6, while the sense primer for Gal-6 was homologous to both mouse and canine Gal-4 exon sequences and the antisense primer for Gal-4 has no match in any data base) (23). Some of the confusion likely comes from the fact that the mouse-specific Gal-6 gene diverged from the more common Gal-4 gene found in a large variety of species (22). Our findings are most consistent with the existence of a canine Gal-4 gene rather than a Gal-6 gene based on gene intron/exon structure and amino acid homology of the coding sequence (see Fig. 1E and supplemental Fig. S4).
We expressed Gal-1, -2, -3, -4, -7, -8, -9N, and -9C as Nterminal tagged GST-fusion proteins in bacteria and glycan preferences were assessed on a synthetic glycan array using a fluorescently tagged anti-GST antibody. Glycan preferences for our canine GST-Gal-1 and -3 were similar to those previously determined for biotinylated human Gal-1 and Gal-3 on the array using galectins at varying concentrations to assess specificity (27): (i) GST-Gal-1 specifically bound sulfated Gal␤4GlcNAc, while GST-Gal-3 did not; (ii) GST-Gal-1 preferred branched N-glycans with terminal Gal␤4GlcNAc, while GST-Gal-3 bound branched N-glycans with terminal fucosecontaining human blood groups A and B; and (iii) GST-Gal-3 binds type 2 polylactosamine regardless of the terminal sugar while GST-Gal-1 prefers terminal Gal␤4GlcNAc. Representative results for GST-Gal-1 and GST-Gal-3 binding on the arrays are presented in Fig. 3. In contrast, we observed a notable difference in glycan preference for the canine GST-Gal-2 when compared with the reported preferences of the human biotinylated Gal-2 on the same array (27). Whereas the human Gal-2 showed significant binding to human blood group A on LacNAc and PL, the canine Gal-2 bound significantly to only biantennary N-glycans with terminal Gal␣1,3 extended Lewis x , with or without core GlcNAc fucosylation.
Glycan preferences for canine GST-Gal-4 on the array were notably different to those previously published for the mouse Gal-4 labeled with Alexa Fluor 488 on an array of biotinylated glycans on streptavidin-coated microtiter plates (28). The mouse Gal-4 showed preference for blood group A and B structures due to binding to the C-terminal CRD and N-terminal CRD, respectively, as well as 3-O-sulfated lactose (28). We found that canine GST-Gal-4 also had preference for blood group A and B although we have not prepared the canine C-terminal CRD and N-terminal CRD separately. Nevertheless, as shown in the supplemental data, GST-Gal-4 binding to sulfated glycans was limited to  ]Gal␤1,4[6-OSO 3 ]Glc␤-R (ranked at 12) which was 10-fold lower than binding to fucosylated trisaccharides.
Human Gal-7 affinity for glycans containing LacNAc was previously studied using isothermal titration microcalorimetry, and was reportedly 6 -11-fold weaker than that observed for bovine Gal-1 and murine Gal-3 (29). More recently, the GST-tagged human Gal-7 was analyzed on the Core H arrays but only at one concentration (200 g/ml) which does not reveal the specificity usually observed at lower concentrations (data is online). Interestingly, we found that the canine GST-Gal-7 preferentially recognized blood group H.
Glycan preferences for canine GST-Gal-8 on the array were similar to those previously published for biotinylated human Gal-8 and the two separate CRDs (30). Whereas human Gal-8 and Gal-8N recognized the same sulfated and sialylated glycans, human Gal-8N did not bind polylactosamine or blood groups antigens; and human Gal-8 and Gal-8C did bind polylactosamine, but not sialylated or sulfated glycans (30). In contrast, we observed that the canine GST-Gal-8 prefers polylactosamine with terminal Neu5Ac␣2,3 (ranked at 1), but not Neu5Ac␣2,6, and it also prefers polylactosamine and branched N-glycans with terminal blood group A (weaker for B). GST-Gal-8 interaction with sulfated glycans was limited to  ]Gal␤1,4[6-OSO 3 ]Glc␤-R (ranked at 31), which was significantly lower than maximal binding on the array.
Glycan preferences for human Gal-9 have not been studied at varying concentrations on the Core H array. Using frontal affinity chromatography, human Gal-9C and Gal-9N CRDs are both reported to bind branched N-glycans and polylactosamine, while human Gal-9N also binds glycolipid-type glycans such as the Forssman pentasaccharide (31). We found that the canine GST-Gal-9N binds preferentially branched N-glycans with terminal blood group A as well as sulfated disaccharides, while GST-Gal-9C prefers sulfated disaccharides but will bind to branched N-glycans with terminal blood group B.
Several galectins appear to have retained an exquisite specificity for human ABO(H) blood group antigens (27,31,33,34). Blood group antigens represent polymorphic antigen structures (35). As a result, it is unlikely that the galectinblood group antigen interactions regulate key processes in the trafficking and localization of cell surface glycoproteins and the initiation of key regulatory events. Indeed, the polymorphic nature of blood group antigens likely arose from unique selective pressures to reduce pathogen invasion (36,37). However, while blood group modification may reduce patho-gen invasion, their presence also eliminates the ability of an individual to develop antibodies against these antigens (35), exposing blood group positive individuals to pathogens that bear cognate antigens. Indeed, blood group positive pathogens are responsible for the induction of anti-blood group antibodies in blood group negative individuals (38,39), suggesting that blood group positive individuals would be uniquely sensitive to infection by blood group positive pathogens. Given the ability of several galectin family members to recognize blood group antigens, recent studies examined whether galectins might uniquely fill this gap in adaptive immunity. Interestingly, Gal-4 and Gal-8 specifically recognize and kill blood group positive E. coli, while failing to recognize or alter the viability of other blood group negative bacteria (40). In this way, Gal-4 and Gal-8 provide protection against blood group positive bacteria regardless of the blood group status of an individual. The ability of several canine galectins to also recognize blood group antigens suggests that these innate immune functions may be conserved, as recently suggested for murine . Future studies will examine these intriguing possibilities.
The possibility still remains that galectins have evolved to play some role in membrane trafficking of transmembrane proteins. There are published data showing that Gal-3, -9, and -1 binding to cell surface proteins such as epidermal growth factor receptor, the glucose transporter Glut-2 and the Ca ϩ2 channel TRPV5, respectively, stabilizes cell surface expression (32,41,42). Gal-3 and galectin-4 have also been implicated in glycan-dependent and raft-dependent sorting, respectively, of apically expressed proteins in polarized epithelial cells (12,13). As MDCK cells are the best characterized model for studying membrane trafficking in polarized epithelial cells, we used the cloned canine galectins to begin to address the potential role of galectins in MUC1 apical targeting in these canine cells. Using the GST-tagged canine galectins in pulldown assays we determined that the small subunit of human MUC1 expressed in MDCK cells binds preferentially to Gal-3 while the large subunit binds preferentially to both Gal-3 and Gal-9N. We also observed that both subunits bound to Gal-1, -4, -7, -8, and -9C but with 2-10-fold lower levels. MUC1 binding to galectin-3 has been previously reported, but this is the first report that MUC1 exhibits significant binding to other galectins, including galectin-9. Yu et al. (17) observed co-immunoprecipitation of endogenous MUC1(large subunit) with galectin-3 from extracts of a human colon cancer cell line (HT29), as well as recombinant galectin-3 binding to MUC1 overexpressed in either MDCK or a human breast epithelial cell line. Argüeso et al. (14) also reported that MUC1 (large subunit) expressed in a human corneal cell line (HCLE) bound to a Gal-3 affinity column in a galactose-dependent manner. However, Ramasamy et al. (15) observed co-immunoprecipitation of galectin-3 and a truncated MUC1 (small subunit only) overexpressed in a ZR-75 breast tumor cell line; and pull-down of the truncated MUC1 with a recombinant GST-tagged human galectin-3 was blocked by mutation of the single N-glycosylation site or by MUC1 expression in glycosylation-defective CHO cells that lack normal N-glycan processing. Therefore, our findings are consistent with studies of MUC1 in other cell types but extend the possibility that MUC1 interaction with additional galectins in MDCK cells are significant and should be studied further.