THE JOURNAL OF BIOLOGICAL CHEMISTRY (e 1987 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 262, No. 30, Issue of October 25, pp. 14753-14759,1967 Printed in U.S.A.

I. Isolation and Characterization of a M, = 110,000 Glycoprotein Localized to the Hepatocyte Bile Canaliculus*

(Received for publication, May 13, 1987)

James K. Petell$$, Mark Diamond$, Wanjin Hong$, Yoram Bujanoverft, Sergio Amarrift, Klaus Pittschielerft, and Darrell Doyle$ From the $Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260 and the 4Colucci Memorial Liver Research Center, International Institute for Infant Nutrition and Gastrointestinal Disease,

A M , =

Children’s Hospital, Buffalo, New York 14222

110.000 glycoprotein, GP 110, was partially purified using wheatgerm agglutinin-Sepharose affin- ity chromatography from a bile canalicular-enriched membrane fraction denoted Nzu of rat liver. This frac- tion was subjected to preparative sodium dodecyl sul- fate-polyacrylamide gel electrophoresis and the M, = 110,000 polypeptide was excised and used as an im- munogen in rabbits. The antisera were found to specif- ically recognize a M, = 110,000 polypeptide, named GP 110, in the Nzu membrane fraction. In isolated hepatocytes, GP 110 was readily accessible to cell sur- face iodination catalyzed by lactoperoxidase at 4 “C and was judged by immunoprecipitation studies to con- tain about 2% of total radioactivity incorporated into externally oriented proteins of the cell. Immunoprecip- itated GP 110 was shown by two-dimensional poly- acrylamide gel electrophoresis to migrate with an ap- proximate PI of 4.9. Indirect immunofluoresence on frozen liver sections demonstrated that GP 110 was primarily localized in the bile canaliculus. In corrobo- rative studies employing subcellular fractionation, it was found that GP 110 was enriched nearly 19-fold in Pz, a plasma membrane fraction primarily derived from the sinusoidal domain, and 44-fold in Nzu. In contrast, only low levels of GP 110 were present in endoplasmic reticulum, mitochrondrial, cytosolic, and nuclear-enriched fractions of liver. The physiological function of GP 110 is as yet unknown; antisera to it did not immunoprecipitate other known bile canalicu- lar proteins of similar molecular weights. GP 110 was found to be extensively glycosylated relative to other known membrane proteins; approximately 33% of the apparent molecular weight appear to be carbohydrate. In agreement, limited removal of N-linked carbohy- drate chains indicated that there are approximately eight chains/GP 110 polypeptide. Neuraminadase treatment of GP 110 resulted in a desialyated M, = 85,000 polypeptide suggesting that the majority of carbohydrate chains on GP 110 are of the complex type.

Rat hepatocytes are polarized epithelial cells with several distinct plasma membrane regions or domains. Examples of such specialized membrane domains include the sinusoidal domain, tight junctions, gap junctions, the bile canaliculus, and the basolateral membrane. Each specific membrane area

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

is characterized by a family of proteins endogeneous to the plasma membrane domain. For example, asialoglycoprotein receptor and insulin receptor are localized to the sinusoidal domain, whereas, dipeptidyl peptidase IV and leucine ami- nopeptidase are restricted to the bile canaliculus (1-6). Ad- ditional insight into the physiological roles of the distinct hepatocyte domains can be obtained by characterization of membrane proteins that are present in that domain.

The overall goal of our laboratory is to identify proteins that reside in distinct hepatocyte domains and to assess their pathway of biogenesis, their turnover behavior in their domain of residence, and ultimately their role in domain function. In this article, we isolate and prepare monospecific antisera to a plasma membrane glycoprotein of M, = 110,000, referred t o as GP 110. By immunocytochemical and cell biological pro- cedures, GP 110 is shown to be primarily localized to the bile canaliculus of the hepatocyte. GP 110 is an extensively gly- cosylated protein with approximately eight N-linked carbo- hydrate chains per polypeptide. In the companion paper (7), we investigate the biosynthesis and turnover of this heavily glycosylated bile canalicular protein.

EXPERIMENTAL PROCEDURES

M a t e r i ~ k ” N a ’ ~ ~ 1 and [35S]metbionine (800 Ci/mmol) were pur- chased from Amersham Corp. and Du Pont-New England Nuclear, respectively. Protein A-Sepharose 4B was obtained from Pharmacia Biotechnology Inc. Triton X-100 was purchased from Accurate Chem- ical (Westbury, NY). N-Glycanase was obtained from Genzyme (Bos- ton, MA). Neuraminidase was bought from Sigma. DMEM’ and fetal calf serum were from Gibco. Collagen was purchased from the Col- lagen Corp. (Palo Alto, CA).

Subcellular Fractionation of Rat Liver and Enzyme Assays-En- riched subcellular fractions representing mitochondrial and lyso- somes, nuclei, endoplasmic reticulum, and cytosol, and two plasma membrane fractions, denoted PZ and NZu, were obtained from livers of adult Buffalo male rats (M. A. Bioproducts, Walkersville, MD) according to the method of Aaronson and Touster (8) as modified by Cook et al. (9). The collected sucrose fractions included the Aaronson and Touster fractions: M and L (mitochondrial and lysosomes), S (cytosol), P4 (endoplasmic reticulum), P2 (sinusoidal plasma mem- brane), and N4 (nuclei) as well as the Cook et al.: Nzu (bile canalicular plasma membrane). The subcellular fractions were immediately as- sayed for organelle marker enzymes and stored at -70 “C to await further processing. The distribution of marker enzymes in these subcellular fractions agreed well with those reported by Aaronson and Touster (8) and Cook et al. (9). Leucine aminopeptidase and dipeptidyl peptidase were assayed using leucine p-nitroanilide and glycyl-L-proline-p-anilide as substrates, respectively, as previously

The abbreviations used are: DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin; HEPES, 4-(2-hydroxyetbyl)- 1-piperazineethanesulfonic acid; WGA, wheat germ agglutinin; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline.

14753

14754 Characterization of GP 11 0

described (2, 10). Other marker enzymes were measured for activity according to published procedures (11-14). Protein determinations were performed by the method of Lowry et al. (15) using bovine serum albumin (BSA) as a standard.

Isolation of G P 11 0 and Immunization-Isolated Nzu plasma mem- branes were extracted in 10 mM HEPES, pH 7.6, 1 mM MgCl,, 1 mM CaC12 (HEPES buffer) containing 2% Triton X-100 in a glass-glass Dounce homogenizer and centrifuged at 120,000 X g for 30 min. The supernatant was incubated with wheat germ agglutinin (WGA) bound to Sepharose beads at 4 "C overnight on an end over end rotator. The following day, WGA-Sepharose beads were placed in a column and were washed extensively with the HEPES buffer containing 0.15 M NaCl and 0.1% Triton X-100 at room temperature to remove non- bound proteins. Bound glycoproteins were specifically eluted from the WGA-Sepharose beads with the washing buffer containing 0.3 M N-acetylglucosamine. Eluted glycoproteins were subjected to prepar- ative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). End strips of the SDS-PAGE were stained for protein with Coomassie Blue while the center portion was stored at 4 "C. Using the stained gel strips as markers, regions of the nonfixed gel containing the M, = 110,000 polypeptide was selectively excised, minced, and incubated in 1: lO diluted PBS containing 0.1% SDS overnight on an end over end rotator a t 4 "C. The diluted PBS buffer containing the protein was concentrated 10-fold and an aliquot was subjected to SDS-PAGE. Approximately 100 pg of purified GP 110 was initially injected in 0.5 ml of complete Freund's adjuvant followed by subsequent injections in incomplete Freund's adjuvant as previ- ously described (16). IgG was prepared from serum through a series of NH4S04 precipitations.

Isolation and Labeling of Hepatocytes-Hepatocytes were isolated from collagenase-perfused rat liver according to Seglen (17) as mod- ified by Petell and Doyle (18). Only preparations of hepatocytes that were greater than 95% viable as judged by exclusion of trypan blue were radioactively labeled. To label the cell surface of hepatocytes, suspensions of hepatocytes were labeled for 30 min with 2 mCi of Na'*'I by lactoperoxidase-catalyzed iodination at 4 "C using the mod- ified procedure of Warren and Doyle (16). Afterwards, hepatocytes were washed by centrifugation in Hank's buffer at 4 "C several times and stored at -70 "C until used. To metabolically label hepatocytes, hepatocytes were cultured for at least 2 h on collagen-coated tissue culture flasks in DMEM containing 10% fetal calf serum and subse- quently incubated in DMEM minus methionine for 60 min. Then, fresh DMEM minus methionine supplemented with 2 mCi of [35S] methionine was added to decanted monolayers. Afterward, hepato- cytes were collected and stored at -70 'C.

Electrophoretic Protocols-Sodium dodecyl sulfate and two-dimen- sional polyacrylamide gel electrophoresis were performed according to the procedures of Laemmli and O'Farrell, respectively (19, 20). The acrylamide concentration of the SDS-PAGE was either 7.5% or 9% as noted. Molecular weights were estimated by comparison to prestained or nonstained standard proteins obtained from Bethesda Research Laboratories (Gaithersburg, MD) or Bio-Rad, respectively. After electrophoresis, polyacrylamide gels were either transblotted to nitromethylcellulose or prepared as follows. In gels to be visualized for protein, the gels were stained with Coomassie Blue. Those gels containing 12sI-labeled proteins were dried and subjected to autora- diography with an intensifying screen and those containing 35S- labeled material were prepared for fluorography and dried as previ- ously described (18). For immunoblots, resulting gels were transblot- ted to nitromethylcellulose paper overnight using a constant voltage of 30 volts (21). Nitromethylcellulose materials were blocked in 5% milk (Carnation) in PBS for 30 min (22). Afterwards, strips were incubated with 5% milk in PBS containing either antibodies against asialoglycoprotein receptor or GP 110 for 3 h on a rotating platform followed by three 10-min washes in milk buffer, Next, strips were incubated with "'I-Protein A for 3 h and subsequently were washed three times for 10 min in milk buffer and subjected to autoradiogra- phy. For immunoquantitation, the resultant autoradiographs were quantitated for radioactivity by a densitometer and expressed per milligram of protein.

itation by extraction in INPTS (50 mM sodium phosphate, pH 7.4, Immunoprecipitation-Samples were prepared for immunoprecip-

0.15 M NaCl, 2% Triton X-100, and 0.5% SDS) containing 1 mM phenylmethylsulfonyl fluoride by hand homogenization and/or soni- cation (two 30-s bursts) at 4 "C. Homogenates were centrifuged at 100,000 X g for 60 min at 4 "C. BSA was added to the supernatant in a final concentration of 1% (INPATS). The mixtures were then incubated with antibodies against GP 110 overnight at 4 "C or for 3

h a t room temperature on an end over end rotator. After incubation, the mixtures were centrifuged for 3 min in a microfuge. The resulting supernatants were incubated with excess Protein A-Sepharose beads preincubated with INPATS either overnight at 4 "C or for 3 h at room temperature. The beads were washed 5 times with INPATS followed by 3 washes in saline/phosphate buffer (INP). Immunopre- cipitated proteins were eluted from Sepharose beads either by boiling in SDS sample buffer (iodinated material) or incubation in 0.2 M glycine, pH 2.5, for 30 min ( [35S]methionine material). Eluted samples so obtained were analyzed by SDS or two-dimensional PAGE as indicated.

Immunofluorescence-Immunofluorescence staining of frozen liver sections was performed using a modified protocol described by Qua- roni (23) as follows. Pieces of perfused adult rat liver were cut into small pieces, immersed in O.C.T. embedding compound (Tissue Tek, Miles Laboratories Inc.) and quickly frozen in liquid nitrogen. 4-6- pm thick sections were cut using a cryostat microtome, placed on glass slides, and allowed to dry at room temperature for 1 h. Liver sections were either immediately prepared for immunofluorescence staining or stored overnight a t -70 "C in a sealed box. Unless other- wise noted all incubations were performed at room temperature. For staining, liver sections were incubated for 1 h at 4 "C in 100 mM sodium phosphate, pH 7.4, containing 0.05% methanol-free formal- dehyde followed by 3 washes with PBS. Sections were then incubated for 1 h at 4 "C in 100 mM glycine in sodium phosphate buffer and then for 30 min in PBS containing 0.2% BSA and washed 3 times with PBS. Next, liver sections were incubated with primary and secondary fluorescein-conjugated antibodies in humidity chambers as follows. Sections were incubated with a 1:lOO dilution of rabbit serum against either GP 110 or asialoglycoprotein receptor in PBS contain- ing 0.2% BSA for 30 min followed by 3 washes with PBS. Afterwards, sections were incubated with a 1:25 dilution of fluorescein isothiocy- anate-conjugated goat anti-rabbit IgG in PBS, 0.2% BSA and washed 3 times with PBS. Liver sections were subsequently mounted in glycerol/PBS, 9:1, and viewed in a microscope with epifluorescence attachment.

N-Glycanase and Neuraminidase Digestions-Following immuno- precipitation of labeled GP 110, aliquots were digested with the appropriate enzyme as follows. According to manufacturers' instruc- tions for N-glycanase, GP 110 was boiled for 3 min in the presence of 0.5% SDS, 0.1 M 2-mercaptoethanol. The sample is diluted with sodium phosphate buffer, pH 8.6, 10 mM 1,lO-phenanthroline con- taining Nonidet P-40 and incubated overnight at 37 "C with N- glycanase (2 units/20 pg of protein). In neuraminidase treatment of GP 110, the sample is diluted in 0.1 M sodium acetate, pH 5.6, and incubated overnight at 37 "C with insoluble neuraminidase (0.5 mil- liunit/pg of protein) as previously described (18).

RESULTS

Preparation of Monospecific Rabbit Antibody against GP 110-A purification scheme was devised to isolate and prepare a monospecific antibody against an abundant M , = 110,000 glycoprotein, GP 110, contained in a bile canalicular-enriched membrane fraction, denoted NZu, of rat liver. The first step of the scheme involved preparation of the Nzu membrane frac- tion from rat liver according to the method of Cook et al. (9). I t was found that the enrichment of 5"nucleotidase in NPu ranged from 50- t o 60-fold over the initial homogenate with low levels of contaminating mitochrondrial and endoplasmic reticulum marker proteins (see Table I). SDS-PAGE of a preparation of Nzu membrane fractions are depicted in Fig. 1 (lane I). The Coomassie Blue-stained polyacrylamide gel shows that GP 110 is 'an abundant protein in the bile cana- licular-enriched fraction. Next, affinity purification of glyco- proteins from NPu membranes was accomplished by WGA- Sepharose chromatography (Fig. 1, lane 2). The eluted glyco- protein fraction contained greater than 95% of the applied GP 110. This fraction was subjected to preparative SDS- PAGE. GP 110 was excised and eluted from the preparative SDS-PAGE (Fig. 1, lane 3) . Purified GP 110 was used as an immunogen for antibody production in rabbits. Immunoblots using the antisera of the immunogenized rabbit detected a single polypeptide of M , = 110,000 in the NPu membrane

Characterization of GP 11 0 14755

TABLE I Enrichment of GP I10 in plasma membrane fractions

A B

Subcellular fraction Relative enrichment"

5"Nucleotidase ASGR GP 110

Pzb 16.8 f 3.8 41 f 3 19 f 6 51.9 f 5.7 1 2 f 1

M and L (2.8)d 44 f 3

0.8 f 0.1 4 . 0 s (3.3)'

4 . 0 0.4 f 0.1 4 . 0

P, (2.4)' -4.0

N, 1.4 f 0.2 2.0 5 0.3 2.0 f 1.0 1.0 f 0.1 4 . 0 <LO

~

5"Nucleotidase enzyme activity or immunoquantitation of asi- aloglycoprotein receptor (ASGR) or GP llO/mg of protein in a subcellular fraction relative to initial homogenate.

Relative enrichment of marker proteins; <O.ld, <0.1', 1.0'. e Relative enrichment of marker proteins; <O.ld, <0.1', 1.3/.

Relative enrichment of cytochrome oxidase. e Relative enrichment of superoxide dismutase. 'Relative enrichment of NADPH-cytochrome c reductase.

cq hiu FIG. 1. Isolation of a n M, = 110,000 glycoprotein from a

bile canalicular-enriched membrane fraction. Aliquots of frac- tions at different stages of GP 110 isolation were analyzed by SDS- PAGE. Lanes: I , Nzu Triton X-100 extract; 2, specifically eluted WGA-bound glycoproteins; 3, M, = 110,000 glycoprotein obtained by preparative SDS-PAGE and 4, molecular weight standards.

fraction (Fig. 2). In contrast, the pre-immune rabbit sera showed no reactivity with this membrane fraction.

Immunoprecipitation Studies with Labeled Hepatocytes- The nature of the antigen recognized by the rabbit sera was characterized by immunoprecipitation studies using isolated hepatocytes which had been cell surface radiolabeled by lac- toperoxidase-catalyzed iodination. In immunoprecipitation studies using a detergent-solubilized cell extract, rabbit anti- GP 110 sera was found to specifically immunoprecipitate a M, = 110,000-labeled polypeptide while no radioactive poly- peptides were recognized by pre-immune sera (Fig. 3). Fur- thermore, it was found that approximately 2% of the total incorporated iodide radioactivity could be immunoprecipi- tated by excess amounts of antisera (Table 11). Therefore, the M, = 110,000 glycoprotein appears to be localized at the hepatocyte surface in an orientation easily accessible to lac- toperoxidase-catalyzed iodination. Evidence that the prepared antisera recognized a single antigen was obtained by subject- ing immunoprecipitated GP 110 to two-dimensional PAGE. As shown in Fig. 3, immunoprecipitated labeled polypeptides migrated as a single heterogenous band with an approximate PI of 4.9. It is not unusual for membrane glycoproteins to show heterogeneity in both size and charge via two-dimen- sional PAGE.

Indirect Immunofluorescence-The immunoprecipitation studies suggested that GP 110 is present on the cell surface

216-

100-

68 -

45 -

FIG. 2. Immunoblot of rabbit antibody against M, = 110,000 glycoprotein. Equal protein aliquots of NPU detergent- solubilized extracts were subjected to SDS-PAGE followed by trans- blotting to nitrocellulose. Nitrocellulose strips were reacted with either pre-immune ( A ) or immune ( B ) antisera and subsequently with radioactive iodinated Protein A and then prepared for autora- diography.

of the plasma membrane of hepatocytes, however, there is no evidence that this glycoprotein resides in a specific hepatocyte domain. To assess its subcellular localization, indirect im- munofluorescence was performed on thin (6 pm) frozen liver sections. Sera directed against GP 110 was localized to a specific region of the liver sections which has the character- istic morphology of bile canalicular structures (Fig. 4). This type of localization for GP 110 is in striking contrast to those liver structures recognized by rabbit sera directed against the asialoglycoprotein receptor or rat hepatic lectin for galactose- terminated glycoproteins, a known sinusoidal membrane pro- tein (Fig. 4). Furthermore, indirect immunofluorescence stud- ies using monoclonal antibodies against two other reported bile canalicular proteins, leucine aminopeptidase and dipep- tidy1 peptidase, showed that identical liver structures repre- senting bile canaliculi were immunofluorescently stained (data not shown).

Subcellular Fractionation of GP 110 with Plasma Membrane Markers-The preceding indirect immunofluorescence stud- ies provided evidence that GP 110 was preferentially localized in the bile canalicular region of the hepatocyte. Sucrose gra- dient fractionation procedures were also used to further char- acterize the localization of GP 110 in hepatocyte membranes. Enriched subcellular fractions were prepared from freshly perfused rat livers using a modification of the Aaronson and Touster (8) method described by Cook et al. (9). The enriched cell fractions were measured for characteristic enzymatic ac- tivities known for different organelles and were quantitated

14756

A. 1 2

Characterization of GP 110

B. I EF-

216-

100-

68-

C

W c3 2

I v, Q m

4.9 1

- iio

45-

FIG. 3. PAGE analysis of GP 110 immunoprecipitated from cell surface-labeled hepatocytes. Hepa- tocytes were surface labeled by lactoperoxidase-catalyzed iodination at 4 “C as described under “Experimental Procedures.” Radioactive GP 110 was immunoprecipitated from Triton X-100 cell extracts and subjected to PAGE and autoradiography. A , SDS-PAGE: lune 1, pre-immune sera; lane 2, immune sera. B, two-dimensional PAGE: first dimension. isoelectric focusine (ZEF): second dimension, SDS-PAGE. The M , = 110,000 glycoprotein has an isoelectric point of 4.9.

L, . I I

TABLE I1 Yield of iodinated M, = 110,000 bile canalicular glycoprotein from cell

surface-labeled hepatocytes Two preparations of hepatocytes, cell surface labeled by lactoper-

oxidase-catalyzed iodination, were extracted in immunoprecipitation buffer and centrifuged as described under “Experimental Procedures.” Samples of labeled supernatant fractions were measured for incor- poration of I2’I into protein as previously described. Aliquots of supernatants were reacted with excess rabbit IgG against GP 110 and analyzed by SDS-PAGE. Gel areas corresponding to GP 110 were excised and counted. Radioactivity in background areas was sub- stracted. The yield of immunoprecipitated GP 110 is represented as the percentage of starting radioactive material.

Sample 1251 Yield

cpm Experiment 1

Triton X-100-solubilized proteins 20,000 100% GP 110 374 1.9%

Experiment 2 Triton X-100-solubilized proteins 20,000 100% GP 100 324 1.6%

for specific membrane proteins, G P 110 and the asialoglyco- protein receptor, by an immunoblot methodology. The en- riched membrane fractions as designated by Aaronson and Touster (8) and Cook et al. (9) represented mitochrondrial and lysosomal, endoplasmic reticulum, cytosolic, nuclear, and two plasma membrane components of the hepatocyte. Similar to 5’-nucleotidase, GP 110 and asialoglycoprotein receptor were greatly enriched (12-44-fold) in the two plasma mem- brane fractions, Nzu and P, (Table I). However, lower levels of G P 110 and asialoglycoprotein receptor were present in other membrane fractions which included the mitochondrial and lysosomal, endoplasmic reticulum, cytosolic, and nuclear fractions. I t is of interest to note that GP 110 like 5”nucleo- tidase was present in 2-3-fold higher levels in NZu, a bile canalicular membrane fraction, than in P,. In contrast, the amount of asialoglycoprotein receptor, a known sinusoidal protein, was about 3-4-fold higher in P, membranes than in N2,, membrane fractions. These results substantiate the im- munofluorescence studies demonstrating that GP 110 is prin- cipally residing in the plasma membrane, and specifically, in

A

8

FIG. 4. Localization of G P 110 and asialoglycoprotein re- ceptor by indirect immunofluorescence. Frozen liver sections (4- 6 pm) were immunofluorescently stained as outlined under “Experi- mental Procedures” using either rabbit antibodies (1 : lOO dilution) directed against GP 110 ( A ) or asialoglycoprotein receptor (B) . White solid arrows indicate bile canalicular structures and white open arrows indicate sinusoidal structures; X 450.

Characterization of GP 11 0 14757

the bile canaliculus of the hepatocyte. Unknown Identity of GP 110-Several hepatocyte mem-

brane enzymes have been reported to be specifically localized in the bile canalicular domain. However, only two of these membrane proteins are possible candidates for G P 110 based on molecular weight and carbohydrate moiety. Dipeptidyl peptidase is a glycoprotein with an apparent molecular weight of 105,000 and leucine aminopeptidase upon limited proteol- ysis may result in a polypeptide of similar molecular weight. To test whether GP 110 is dipeptidyl peptidase or leucine aminopeptidase, aliquots of detergent-solubilized NSu mem- branes were incubated with sufficient antisera to immunopre- cipitate all G P 110 polypeptides as judged by immunoblot quantitation. As shown in Table 111, the bulk of dipeptidyl peptidase and leucine aminopeptidase enzymatic activities were present in the supernatant of the immunoprecipitated fraction. Pre-immune sera did not immunoprecipitate either dipeptidyl peptidase and leucine aminopeptidase enzyme ac- tivities or G P 110. Therefore, the identity of G P 110 cannot yet be assigned to a known enzymatic activity and its function is yet to be elucidated.

Degree of GP 11 0 Glycosylation-The preferential binding of G P 110 to WGA and concanavalin A coupled to Sepharose beads together with periodic acid-Schiff staining demon- strates its glycoprotein nature (data not shown). In order to gain insight into the nature of the carbohydrate moieties of G P 110 we subjected immunoprecipitated labeled G P 110 to two treatments. First, G P 110 was treated with N-glycanase which removes N-linked carbohydrate chains by cleaving between N-acetylglucosamine of the sugar chain and an as- parginine residue of the polypeptide. The apparent molecular weight of G P 110 was reduced to approximately 59,000 by this treatment (data not shown). This result means that the carbohydrate moiety of G P 110 represents a t least 45% of its apparent molecular weight as judged by SDS-PAGE. Next, G P 110 was incubated with neuraminadase to remove termi- nal sialic acid residues from complex carbohydrate chains. G P 110 so treated was observed to decrease in size by M , = 25,000 indicative of a complex carbohydrate structure (data not shown). Furthermore, it appears that sialylation of the car- bohydrate chains of GP 110 artifactually accounts for a t least 50% of the carbohydrate molecular weight on SDS-PAGE. Thus, assuming an average molecular weight of 3,000 per asialocarbohydrate chain, we estimate there to be as many as eight or nine N-linked chains per G P 110 molecule and that approximately 33% of the molecular weight of GP 110 is carbohydrate.

To more precisely determine the number of N-linked chains per GP 110 polypeptide, isolated hepatocytes were metaboli- cally labeled in culture with ["S]methionine for 5 h. Then, radiolabeled G P 110 was purified by immunoprecipitation and

TABLE I11 Characterization of bile canalicular proteins immunoprecipitated by

anti-GP 110 In this representative experiment, an aliquot of Nau membranes

solubilized in immunoprecipitation buffer was reacted with an excess amount of rabbit IgG against GP 110. After immunoprecipitation, the initial extract and the supernatant were measured for leucine aminopeptidase (LAP) and dipeptyl peptidase (DPP) enzyme activ- ities and immunoquantitated for GP 110. Greater than 90% of G P 110 was present in the immunoprecipitate as assessed by immuno- blots.

IgG Source % Remaining in supernatant

LAP DPP GP 110

Pre-immune 100 100 100 Anti-GP 110 100 100 8

A. I E F + W W

2 cn a

4

8. IEF- W c3 4 a

5.8 I

4.9 I

4.5 4.9 I I

-110

- 75

-iio

- 59

FIG. 5. Two-dimensional analysis of GP 110 N-linked car- bohydrate chains. Isolated hepatocytes were metabolically labeled with [:"SS]methionine for 3 h, extracted, and reacted with rabbit anti- GP 110 IgG. Immunoprecipitated GP 110 was subjected to limited treatment with N-glycanase followed by two-dimensional PAGE and autoradiography. A, GP 110; B, N-glycanase-treated GP 110. IEF, isoelectric focusing.

subjected to limited N-glycanase treatment followed by two- dimensional PAGE and fluorography. As shown in Fig. 5, in the absence of N-glycanase two distinct labeled polypeptides were immunoprecipitated; M , = 110,000 polypeptide with a PI of 4.9 and M , = 75,000 with a PI of 5.8. The precise relationship of these polypeptides will be detailed in the companion manuscript (7). Upon limited treatment with N- glycanase, eight distinct forms can be identified with molec- ular weights ranging from 59,000 to 110,000. The M , = 59,000 polypeptide was observed to have a PI of 5.3. Therefore, from these data it is concluded that GP 110 contains as many as eight N-linked carbohydrate chains per polypeptide.

DISCUSSION

Considerable progress has been made in recent years in the identification of plasma membrane proteins that are localized in specific domains of hepatocyte (1-6, 9, 24-39). Evidence for the localization of a given membrane protein in a specific hepatocyte domain has been based on catalytic, histological, and/or immunological approaches (1-6,9,24-39). In the case of some proteins their resident domain has been controversial. However, certain membrane proteins have been rigorously shown to be restricted to a specific domain. Probably the most well characterized hepatocyte membrane protein is an abundant sinusoidal glycoprotein responsible for removing those serum glycoproteins with their penultimate galactose

14758 Characterization of GP 110

residues exposed; the hepatic binding lectin or asialoglycopro- tein receptor (1, 3-6). Two other membrane glycoproteins, leucine aminopeptidase and dipeptidyl peptidase, have been convincingly shown by several investigators including our laboratory to be localized in the bile canaliculus (2-5, 26, 29, 39). The goal of this and ensuing investigations has been to identify other membrane proteins and ascertain their local- ization in a specific domain by immunological and biochemical approaches. Valuable information on the physiological role of specific hepatocyte domains might be ultimately deduced from the protein constituents in the domain. Furthermore, assign- ment of sets of proteins to different domains should in the future aid in unraveling the mechanism of biogenesis of the hepatocyte domains both in differentiated adult cells and during development.

In the past our laboratory has used a sucrose gradient protocol based on the original method of Aaronson and Tous- ter (8) for obtaining a plasma membrane fraction derived from the bile canaliculus referred to as NZu. Here, we used this procedure to prepare a NZu fraction which was enriched 50-60-fold in 5’-nucleotidase activity. AM, = 110,000 peptide observed to be abundant in Nzu membranes was selected for study. Based on preliminary studies showing that the M, = 110,000 protein bound specifically to lectin-Sepharose col- umns, a simple purification scheme was devised to isolate the M , = 110,000 glycoprotein referred to as GP 110. Polyclonal rabbit sera directed against purified GP 110 was shown to be monospecific by an immunoblot protocol. With the monspe- cific antisera in hand we were able to examine certain bio- chemical properties of GP 110.

Immunoprecipitation studies using cell surface-radiolabeled hepatocytes revealed that GP 110 was an externally oriented membrane protein and similar to the asialoglycoprotein re- ceptor was readily accessible to lactoperoxidase-catalyzed io- dination. GP 110 was observed to be acidic in nature with a PI of 4.9. The acidic nature and heterogeneity of G P 110 is in part due to its numerous N-linked complex carbohydrate chains which contain terminal sialic acid residues. G P 110’s carbohydrate moieties account for 45% of its apparent molec- ular weight as assessed by SDS-PAGE. It should be noted that the degree of sialylation may result in an overestimation of the actual carbohydrate size. In view of this, we estimated that there are approximately eight N-linked chains per poly- peptide assuming that the average molecular weight of desi- alylated carbohydrate chains is -3000. This estimation of N- linked chains was supported by a limited removal of these chains by N-glycanase. Additional support for eight carbo- hydrate chains per polypeptide has been obtained by Hong and Doyle (40) who determined the derived amino acid se- quences of GP 110 and found there to be eight asparagine acceptor sites for N-linked carbohydrate attachment. I t is clear that relative to other plasma membrane proteins G P 110 is one of the more extensively glycosylated proteins. In the companion paper (7 ) , the time frame of biosynthesis of the heavily glycosylated GP 110 is compared relative to asi- aloglycoprotein receptor which has only one to two complex carbohydrate chains (3).

In this report we demonstrate by an indirect immunofluo- rescence assay on frozen liver sections that GP 110 is princi- pally concentrated in the bile canaliculus of the hepatocyte. Additional evidence for its localization in the plasma mem- brane, and specifically the bile canaliculus, was obtained by a classical biochemical/cell biological approach. Utilizing a su- crose gradient methodology the levels of GP 110 were assessed in various subcellular fractions. G P 110 was enriched 44- and 19-fold in two plasma membrane fractions, NZu and Pz, re-

spectively, which had low amounts of other contaminating cellular organelles. The enrichment of asialoglycoprotein re- ceptor was markedly different in Nzu and Pz membranes with approximately a 4-fold higher receptor level in the latter fraction. This finding is not surprising but instead supportive of the assumption that Pz membranes are derived primarily from the sinusoidal domain while NPu membranes originate from the bile canaliculus. Indeed, the biochemical data con- vincingly support the immunofluorescence study to demon- strate that GP 110 is primarily restricted to the bile canalic- ulus.

In the past, several membrane proteins, primarily enzymes, have been implicated to be localized in the bile canalicular domain. The majority of these reports were based on the catalytic nature of the protein as followed in subcellular fractionation or in histological studies. More recent methods based on immunological techniques have proven more reliable for vigorous localization studies. Putative bile canalicular proteins with known catalytic activity include leucine ami- nopeptidase, dipeptidyl peptidase, y-glutamyltransferase, 5’- nucleotidase, alkaline phosphatase, M$+-ATPase, and alka- line phosphodiesterase (2-5, 24-39). Only leucine aminopep- tidase and dipeptidyl peptidase would be likely candidates to be G P 110. Both are membrane glycoproteins with similar molecular weights and have been shown by immunological criteria to be localized in the bile canaliculus. However, nei- ther of these enzymatic activities were immunoprecipitated by antisera to GP 110 while all authentic GP 110 molecules were. Furthermore, the size of the carbohydrate moiety of GP 110 was in far excess of that reported for either enzyme. Thus, GP 110 does not yet have a known catalytic activity or function in the bile canaliculus.

In addition to membrane enzymes, recent studies have demonstrated the presence of other proteins with unknown enzymatic activities or functions in the bile canaliculus. Ock- lind et al. (41, 42) have identified a bile canalicular glycopro- tein with a molecular weight of 105,000 which is involved in rat hepatocyte intercellular adhesion, referred to as cell adhe- sion molecule (cell-CAM 105). Cell CAM-105 was also found to be present in the apical domain of intestine epithelial cells and kidney tubules. Similarly, Hixson et al. (43, 44) have recently reported a cell-CAM 105 that is present in the apical domains of liver, intestine, and kidney tissues. Likewise, G P 110 was detected by immunocytochemical methodology in apical regions of kidney and intestine tissue as well as liver.* Cell-CAM 105 has a PI of 4.6-4.9 and upon treatment with N-glycanase resulted in a M , = 37,000 polypeptide. Hubbard and colleagues (3-5) utilizing monoclonal antibodies prepared against rat liver plasma membranes identified a bile canali- cular protein, called HA 4, with a molecular weight of 105,000 which did not immunoprecipitate any known membrane en- zyme. Analogous to GP 110 and CAM’s, HA 4 was extensively glycosylated predominantly consisting of N-linked complex carbohydrate moieties (3-5). In contrast, immunofluorescence revealed that HA 4 was present in intestinal epithelial cells but not kidney tubules. Lastly, in a preliminary report, Land- mann et al. (45) have isolated an organic anion transport protein which is concentrated in the apical region of liver, ileum, and kidney epithelial cells. This organic anion trans- porter has a molecular weight of 100,000 and upon chemical deglycosylation was reduced to 48,000. The characteristics of GP 110 bear a striking resemblance in size, charge, and glycosylation to CAM 105, HA 4, and organic anion trans- porter. Based on their structural and morphological similari-

J. K. Petell, W. Hong, and D. Doyle, unpublished observation.

Characterization of GP 110 14759

ties, it is probable that these four glycoproteins are the Same 14. Arketeign, c. L. M. (1976) J. Clin. Chem. C h . Biochem. 1 4 ,

glycoprotein. the physiological that this unique 15. Lowry, 0. H., hsebrough, N. J., Farr, A. L., and Randall, R. J. 155-169

glycoprotein plays as a cell adhesion molecule and/or bile acid (1951) J. Biol. Chem. 193 , 265-275 transporter, or other bile canalicular function remains to be 16. Warren, R., and Doyle, D. (1981) J. Biol. Chem. 2 5 6 , 1346-1355 conclusively determined. 17. Seglen, P. 0. (1976) Methods Cell Biol. 13 , 29-83

The uniqueness of GP 110 with respect to its extensive 18. Petell, J. K., and Doyle, D. (1985) Arch. Bimhem. BiophYs. 2 4 1 ,

carbohydrate structure raises interesting questions concern- 19. Laemmli, u. K. (1970) Nature 227, 680-685 ing its biosynthesis and eventual turnover. In the accompa- 20. O'Farrell, P. H. (1975) J. BWl. Chem. 250,4007-4021 nying paper (7), we investigated the time of biosynthesis of 21. Towbin, H., Stahelin, T., and Gordon, J . (1979) Proc. Natl. Acad. GP 110 relative to asialoglycoprotein receptor, a sinusoidal Sci. U. S. A. 76,4350-4354

glycoprotein with a small number of carbohydrate chains. It 22. Johnson, D. A., Gautsch, J. W., Sportman, J. R., and Elder, J. H.

may be postulated that bile canalicular proteins have specific 23. Quaroni, A. (198.5) J. Cell ~ i o l . 100, 1601-1610 (1984) Gene Anal. Tech. 1 , 3-8

internal trafficking signals that direct these proteins to their 24. DeBroe, M. E., Roels, F., Nouwen, E. J. Claey, S. J., and Nieme, domain of residence. Alternatively, their peptide sequence R. J. (1985) Hepatology (Baltimore), 5 , 118-128 may act to dictate their localization. Clearly, additional infor- 25. Scharschmidt, B. F., and Keefe, E. B. (1981) Biochim. Biophys.

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