Vol. 170, No. 3, 1990 August
BIOCHEMICAL
16, 1990
THYROID
HORMONE
BINDING
SITE BINDING
Hitomi
Kimural,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1319-1324
PROTEIN CONTAINS GLYCOSYLATION PROTEIN ACTIVITY
Robert Noiva2, Takemitsu
Ryuya Horiuch?,
Mizunaga3, Kiyoshi Yamauchi4,
Sheue-Yann Cheng6 and William
‘Department
of Biochemistry
J. Lermarz”
and Cell Biology,
SUNY at Stony Brook, Stony Brook, NY 11794-5215 2Department of Biochemistry and Molecular Biology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030 3Department University
of Agricultural
of Tokyo, Bunkyo-ku,
4Faculty of Science, Shizuoka University, ‘Institute
of Endocrinology,
%aboratory Received
July
9,
of Molecular
Chemistry, Tokyo 113, Japan
836 Ohya, Shizuoka 422, Japan
Gunma Univ., Maebashi, Gunma 371, Japan Biology, N.C.I., N.I.H.,
Bethesda, MD
20892
1990
Several lines of evidence provided by other workers indicate that within the same species thyroid hormone binding protein, the b-subunit of prolyl hydroxylase, and protein disulfide isomerase are the same protein. We sought to determine if glycosylation site binding protein, a lumenal protein of the endoplasmic reticulum, also has the same primary structure. To accomplish this the level of glycosylation site binding protein (GSBP) activity, measured by photolabeling with a glycosylation site peptide probe, was carried out in preparations of 3T3 cells and in E- &j transformed with human thyroid hormone binding protein cDNA. The results strongly support the idea that GSBP is identical to these other lumenal proteins of the endoplasmic reticulum. o 1990Academic Press, Inc. In an earlier effort to identify components of oligosaccharyl transferase, the enzyme involved in the process of N-glycosylation, we developed a lZI-labeled photoaffinity probe Using this photoreactive, containing the acceptor tripeptide sequence, Asn-Lys-Thr. radiolabeled peptide, we identified a 55-57 kDa protein (named Glycosylation Site Binding Protein,
GSBP) that was found to be a resident protein
of the lumen of hen oviduct
microsames (1). Subsequently, a protein of similar molecular weight was detected in microsomal preparations from a variety of other hen tissues, as well as in microsames from other higher eukaryotes (2). Using antibody prepared against the hen oviduct GSBP, a xgtll
expression library was screened, a clone expressing GSBP was isolated, and the
‘To whom correspondence should be addressed. 0006-291X&O
1319
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
cDNA was sequenced (3). Comparison of the deduced amino acid sequence of this hen oviduct protein with other sequences revealed that GSBP had 82-88% sequence similarity to rat liver protein disulfide isomerase (PDI), human thyroid hormone binding protein (THBP) and the human p-subunit of prolyl hydroxylase (B-PH), all three of which had been earlier cloned and sequenced by three independent
research groups (4, 5, 6). In
fact, evidence has been published that within a single species PDI, THBP
and p-PH are
the same polypeptide (7). In the current study we have tested the hypothesis that GSBP is identical other proteins by determining
if introduction
of human THBP
to these
cDNA into 3T3 cells (8)
leads to enhanced GSBP activity as assessed by photoaffinity probe labeling. Similarly, we have asked if AE coli cells transformed with the human THBP cDNA express GSBP activity. The positive results of these experiments in both eukaryotic and prokaryotic cell preparations
indicate
support that THBP,
that THBP
contains GSBP activity, and thereby provide further
PDI, 8-PH and GSBP are the same lumenal protein. MATERIALS
AND
METHODS
Materials: The acceptor tripeptide containing a photoreactive group and a carboxy terminus derivatized as an amido group, Asn-Lys-(N-E-p-azidobenzoyl)Thr-NH,, was synthesized as reviously described (9) and then it was derivatized (9) at its amino terminus with 525I-labeled Bolton-Hunter reagent (2000 Ci/mmole) purchased from Amersham, Inc. Methods: NIH 3T3 cells were infected with virus, with or without human THBP cDNA (pHTBr), and then collected by centrifugation (8). Expression of THBP was measured in a cell free preparation as previously described using ‘xl-labeled thyroid hormone binding protein complex (8). To study GSBP activity, 200 ~1 of packed cells (approximately 8 x lo6 cells) were suspended in a final volume of 1.0 ml of 50 mM Tris-HCl, pH 7.5, to which Triton X100 was added to a final concentration of 0.1% (v/v). The cells were sonicated by a Branson cell disruptor 185 at a number 5 setting for 5 seconds, followed by incubation on ice for 20 seconds The sonication and chilling steps were repeated 6 times. The resulting suspension was centrifuged in a Beckman ultracentrifuge Ty65 rotor at 233,000 x g for 1 hr. at 4°C. The supernate and resuspended pellet fractions were photolabeled as described below. Alternatively, 500 ~1 of packed cells (approximately 2 x lo7 cells, 9.5 mg of total protein) were suspended in a final volume of 1.0 ml containing 50 mM Tris-HCl, pH 7.5, 154 mM sucrose, 25 mM NaCl, 1 mM EDTA, 10 mM MnCl,, 10 mM ,+mercaptoethanol (TSNE buffer) with the following protease inhibitors: leupeptin (1.0 pg/ml), antipain (2.0 pg/ml), benzamidine (10.0 pg/ml) and aprotinin (1.0 pg/ml). The cells were sonicated under the conditions described above, but for 3 second intervals followed by incubation on ice for 30 seconds. Sonication and chilling steps were repeated 5 times. The suspension was centrifuged in a Beckman Microfuge 11 at 14,000 x g for 15 min. at 4°C. After the supernate was removed, the pellet was resuspended in an equal volume of buffer by brief (l-2 seconds) sonication. A clone containing the cDNA of human THBP was isolated from a human placenta $tll cDNA library (Clontech, Palo Alto, CA) using as probe a previously described (11) P-labeled 0.3 kbp EcoRI-Pvull fragment of bovine THBP cDNA. The resultant cloned DNA was subcloned in a plasmid pUC19. Then this 1.96 kbp DNA fragment (pT,BP), containing the coding region of the THBP lacking the coding region for signal peptide, 1320
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
was subcloned into the EcoRI-PstI site of plasmid pTrp781, which contained the a promoter, tetracycline resistance gene and plasmid replicon origin (12). E. coli MM294 was transformed by this recombinant plasmid. Transformed E. coli MM294 with or without the plasmid (pT,BP) was grown in 2xTy media (16 g/l Bacto tryptone, 10 g/l Bacto yeast extract, 5 g/l NaCl) to an absorbance of 0.4 at 550 nm. Two ml of the culture were transferred to 50 ml of minimum media (6 g/l Na,HPO,, 3 g/l IQ&PO,, 0.5 g/l NaCl, 1 g/l NH&l, 1% (w/v) glucose, 0.2% Casamino acids) containing tetracycline (12.5 pg/ml) and grown to the same absorbance. Expression of THBP was induced by addition of 3-indoleacrylic acid at a final concentration 25 pg/ml. After cultivation for an additional 4 hours, the cells were harvested by centrifugation at 3,000 rpm for 20 min. at 4”C, washed with 1.50 mM NaCl, then suspended in 1 ml of 10 mM Tris-HCl, pH 8.0 containing 10% sucrose, 200 mM NaCl, 1 mM EDTA and protease inhibitors. The cells were ruptured by sonication as described above for 10 seconds and incubated on ice for 30 seconds. The sonication and chilling procedure was repeated 5 times. Photolabeling of proteins with the ‘?-labeled azidopeptide was carried out as previously described (10). Supernate or membrane proteins from 3T3 cells or from E. &i in a final volume of 100 ~1 were incubated with 1 nmole of azidopeptide in the dark for 20 min. then irradiated at 254 nm for 2 min. After addition of 100 ~1 of SDS sample buffer, the sample was heated at 100°C for 2 min. and then subjected to SDS/PAGE followed by autoradiography. Labeling of the 57 kDa protein was quantitated using a densitometer. To measure the level of the lumenal marker P-glucuronidase in preparations of control and transformed 3T3 cell extracts, hydrolysis of 4-methylumbelliferyl glucuronide (UG) was determined. UG (5.0 mM) in 100 mM sodium citrate, pH 5.2, was incubated with the appropriate cell fraction at 37°C for 10 or 20 min. After termination of the reaction by addition of 0.1 N NaOH, fluorescence was measured by using a Perkin-Elmer LS3B fluorescence spectrometer at an excitation wavelength of 365 nm and an emission wavelength of 460 nm. RESULTS The discovery that hen oviduct GSBP had high sequence similarity to PDI, B-PH and THBP in various species (3) coupled with the finding that PDI, 8-PH and THBP were identical
(5,6,13), led us to consider if GSBP also was the same polypeptide.
The
approach taken was to carry out determinations of GSBP activity in both eukaryote and prokaryote cells expressing the THBP gene product. Our initial efforts focused on NIH 3113 cells transformed with human THBP cDNA. As previously reported (S), we found that the transformed cells contained a 55-57 kDa protein that bound ‘251-labeled T, and could be precipitated with a polyclonal antibody. Consistent with the results of earlier findings (8), the level of THBP was found to be approximately 4.5 times greater in cells transformed with the THBP cDNA than it was in control cells (Fig. 1A). Thus, introduction of the exogenous THBP cDNA markedly increased the level of THBP over the endogenous level found in the control cells. As expected if the THBP cDNA encodes for GSBP, when the ability to bind the glycosylation site photoprobe was assessed an approximately 5.5 fold increase in GSBP was detected (Fig. 1B). In a second experiment carried out in the absence of detergent, it was established that in ruptured 3T3 cells the 57 kDa labeled protein was localized to the 1321
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
,
RESEARCH
INSERT+ ’
SUP. --==mm
MEM.
COMMUNICATIONS
. kz’ 2%
INSERTSUP.
I MEM.
Figure
1. Expression of THBP and GSBP Activity in Transformed 3T3 Cells. A. Control cells (&ane 1) and transformedcells(Lane 2) were cooled to 15°Cand incubated with 0.5 nM ’ I-BrAc T3 for 1 hr. Cells were washed,extracted with 3 mM CHAPS and immunoprecipitatedwith polyclonal antibody 3632. The pellet was analyzed for labeled proteins after SDS/PAGE. El. Packedcells (200 ~1)were extracted with 0.1% Triton X100, sonicationand ultracentrifugation at 233,000x g for 1 hr. Supernatewas incubated with 1.0 nmole ‘251-azidopeptide probe at room temperaturefor 20 min., photolysedand electrophoresedon 7.5%-SDS/PAGE. In both A and B, ?labeled proteins were detected by autoradiographyand the relative level was measuredby densitometty. Figure 2. Distribution of GSBP Activity in Membrane and Supernatant Fractions of Transformed and Control Cells. Transformedcells (Lanes 1-6) or control cells (Lanes
8-13) were sonicatedin TSNE buffer and centrifugedat 14,000x g for 15 min. After the membranepellet was suspendedin the buffer, both the supernateand the membrane fractions were incubated with 1.0 nmole ‘=I-azidopeptide probe for 20 min. at room temperature, and photolysed. Both fractions were then subjectedto 7.5%-SDS/PAGE followed by autoradiography. The profile obtained with rat liver microsomeis shownin Lane 7.
membrane fraction, rather than the supernate fraction (Fig. 2) and that its level was markedly increased in the transformed cells when compared to the control celIs. Indeed, densitometry measurements, shown in Fig. 3, revealed that there was a linear response in
6Ins-
04 Firmre 3. Quantitation of the Level of %photolabeled Proteins in Transformed and Control 3T3 Cells. The relative levels of photolabeled proteins were quantified by
scanningof the autoradiogramsshownin Fig. 2. The level of the ‘ZI-photolabeledprotein in transformedcells (0) was 4.5-fold higher than that in control cells (0). Figure 4.
p-Glucuronidase
Activity in Microsomes
From Transformed
and Control
313
Cells. Cellswere sonicatedin buffer without Triton X-100, and then incubatedwith 5.0 mM UG in 100mM citrate buffer, pH 5.2 at 37°C. The reactionwasstoppedby addition of 0.1 N NaOH. Fluorescenceof UG from transformedcells (A) and control cells (A) at 10min. incubation or transformedcells (0) and control cells (0) at 20 min. incubation was measuredby Perkin-Elmer IS-3B FluorescenceSpectrometer. I322
vol.
170.
No.
3, 1990
BIOCHEMICAL
g "i 2
AND
E coli INSERT+ L-2 0s
2
BIOPHYSICAL
I5 coli INSERT10 0 k3 -
3
4
5
RESEARCH
COMMUNICATIONS
E. coli ALONE '0 Oa
6
7
Fipure 5. Expression of GSBP Activity in E. coli. The total protein fraction from E. coJ transformedwith expressionvector containingTHBP cDNA (Lanes2,3), expressionvector alone (Lanes 45) or E. coli alone (Lanes 6,7) were incubated with 1.0 mnole ‘=Iazidopeptideprobe for 20 min. at room temperature,photolysedand analyzed by 7.5% SDS/PAGE. Lane 1 showsphotolabeledrat liver microsomes.
labeling with the azidopeptide increase in photolabeling
in the range of 20-100 pg of protein, and that the overall
in the cells transformed was 4.5 fold, in excellent agreement with
the observed increase in the level of THBP. Because it seemed possible, albeit unlikely, that transformation of human THBP cDNA into 3T3 cells induced a general increase in the level of microsomal proteins, we determined
the level of the microsomal
marker
enzyme, @-glucuronidase,
in both
transformed and control 3T3 cells. As shown in Fig. 4, under conditions where microsomal enzyme activity was linearly dependent on the amount of protein and time of incubation
there was no difference in the level of this microsomal
control and transformed
marker
enyzme in
cells.
Finally, we examined E. coli cells that had been transformed with the human TJXBP cDNA.
As shown in Fig. 5, the THBP transformed E. coli expressed a 55-57 kDa protein
that binds the photoaffinity probe, whereas neither E. coli containing coli alone expressed this protein.
only vector nor E.
The results of these studies allow one to expand the list of properties of the human 57 kDa protein found in the lumen of the endoplasmic reticulum to include binding of a peptide containing a N-glycosylation site. Thus, it appears that a single 55-57 kDa lumenal protein functions: 1) in the form of a homodimer that functions to scramble disulfide bonds (PDI); 2) as the B component of the CY& tetramer that hydroxylates prolyl residues (B-PH);
3) as a binding protein that recognizes elements in the structure of thyroid hormone (THBP); and 4) as a binding protein that recognizes N-glycosylation site peptides (GSBP). The possibility that this apparently multifunctional binding protein plays a role in protein maturation in the lumen of the endoplasmic reticulum is intriguing. ACKNOWLEDGMENTS:
and NIH Postdoctoral
This work was supported by NIH Grant GM 33185 to W.J.L., Fellowship GM12628
to R.N.
1323
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOFHYSICAL
RESEARCH
COMMUNICATIONS
REFERENCES 1.
2. 3. 4.
Welply, J.K., Shenbagamurthi, P., Naider, F., Park, H.R., and Lennarz, W.J. (1985) J. Biol. Chem. 260, 6459-6465. Kaplan, H.A., Welply, J.K., and Lennarz, W.J. (1987) Biochim. Biophys. Acta 906, 161-173. Geetha-Habib, M., Noiva, R., Kaplan, H.A., and Lennarz, W.J. (1988) Cell 54, 1053-1060. Edman, J.C., Ellis, L., Blancher, R.W., Roth, R.A., and Rutter, W.J. (1985) Nature 317, 267-270.
5. 6. 7. 8. 9. 10. 11. 12. 13.
Cheng, S., Gong, Q., Parkinson, D., Robinson, E.A., Appella, E., Merlino, G.T., and Pastan, I. (1987) J. Biol. Chem. 262, 11221-11227. Pihlajaniemi, T., Helaakoski, T., Tasanen, K., Myllyla, R., Huhrala, M-L., Koivu, J., and Kivirikko, K.I. (1987) EMBO J. 6, 643-649. Parkkonen, T., Kivirikko, K.I., and Pihlajaniemi, T. (1988) Biochem. J. 256, 10051011. Kato, H., Velu, T., and Cheng, S-Y. (1989) Biochem. Biophys. Res. Commun. 164, 238-244. Kaplan, H.A., Naider, F., and Lennarz, W.J. (1988) J. Biol. Chem. 263, 7814-7820. Welply, J.K., Kaplan, H.A., Shenbagamurthi, P., Naider, F., and Lennarz, W.J. (1986) Arch. Biochem. Biophys. 246, 808-819. Yamauchi, K., Yamamoto, T., Hayashi, H., Koya, S., Takikawa, H., Toyoshima, T., and Horiuchi, R. (1987) Biochem. Biophys. Res. Commun. 146, 1485-1492. Kurosawa, T., Sasada, R., Iwane, M., and Igarashi, K. (1987) Nucl. Acid Res. 11, 3077-3085. Koivu, J., MyIlyia, R., Helaakoski, T., Pihlajaniemi, T., Tasanen, K., and Kivirikko, K.I. (1987) J. Biol. Chem. 262, 64476449.
1324
BIOCHEMICAL
16, 1990
THYROID
HORMONE
BINDING
SITE BINDING
Hitomi
Kimural,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1319-1324
PROTEIN CONTAINS GLYCOSYLATION PROTEIN ACTIVITY
Robert Noiva2, Takemitsu
Ryuya Horiuch?,
Mizunaga3, Kiyoshi Yamauchi4,
Sheue-Yann Cheng6 and William
‘Department
of Biochemistry
J. Lermarz”
and Cell Biology,
SUNY at Stony Brook, Stony Brook, NY 11794-5215 2Department of Biochemistry and Molecular Biology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030 3Department University
of Agricultural
of Tokyo, Bunkyo-ku,
4Faculty of Science, Shizuoka University, ‘Institute
of Endocrinology,
%aboratory Received
July
9,
of Molecular
Chemistry, Tokyo 113, Japan
836 Ohya, Shizuoka 422, Japan
Gunma Univ., Maebashi, Gunma 371, Japan Biology, N.C.I., N.I.H.,
Bethesda, MD
20892
1990
Several lines of evidence provided by other workers indicate that within the same species thyroid hormone binding protein, the b-subunit of prolyl hydroxylase, and protein disulfide isomerase are the same protein. We sought to determine if glycosylation site binding protein, a lumenal protein of the endoplasmic reticulum, also has the same primary structure. To accomplish this the level of glycosylation site binding protein (GSBP) activity, measured by photolabeling with a glycosylation site peptide probe, was carried out in preparations of 3T3 cells and in E- &j transformed with human thyroid hormone binding protein cDNA. The results strongly support the idea that GSBP is identical to these other lumenal proteins of the endoplasmic reticulum. o 1990Academic Press, Inc. In an earlier effort to identify components of oligosaccharyl transferase, the enzyme involved in the process of N-glycosylation, we developed a lZI-labeled photoaffinity probe Using this photoreactive, containing the acceptor tripeptide sequence, Asn-Lys-Thr. radiolabeled peptide, we identified a 55-57 kDa protein (named Glycosylation Site Binding Protein,
GSBP) that was found to be a resident protein
of the lumen of hen oviduct
microsames (1). Subsequently, a protein of similar molecular weight was detected in microsomal preparations from a variety of other hen tissues, as well as in microsames from other higher eukaryotes (2). Using antibody prepared against the hen oviduct GSBP, a xgtll
expression library was screened, a clone expressing GSBP was isolated, and the
‘To whom correspondence should be addressed. 0006-291X&O
1319
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
cDNA was sequenced (3). Comparison of the deduced amino acid sequence of this hen oviduct protein with other sequences revealed that GSBP had 82-88% sequence similarity to rat liver protein disulfide isomerase (PDI), human thyroid hormone binding protein (THBP) and the human p-subunit of prolyl hydroxylase (B-PH), all three of which had been earlier cloned and sequenced by three independent
research groups (4, 5, 6). In
fact, evidence has been published that within a single species PDI, THBP
and p-PH are
the same polypeptide (7). In the current study we have tested the hypothesis that GSBP is identical other proteins by determining
if introduction
of human THBP
to these
cDNA into 3T3 cells (8)
leads to enhanced GSBP activity as assessed by photoaffinity probe labeling. Similarly, we have asked if AE coli cells transformed with the human THBP cDNA express GSBP activity. The positive results of these experiments in both eukaryotic and prokaryotic cell preparations
indicate
support that THBP,
that THBP
contains GSBP activity, and thereby provide further
PDI, 8-PH and GSBP are the same lumenal protein. MATERIALS
AND
METHODS
Materials: The acceptor tripeptide containing a photoreactive group and a carboxy terminus derivatized as an amido group, Asn-Lys-(N-E-p-azidobenzoyl)Thr-NH,, was synthesized as reviously described (9) and then it was derivatized (9) at its amino terminus with 525I-labeled Bolton-Hunter reagent (2000 Ci/mmole) purchased from Amersham, Inc. Methods: NIH 3T3 cells were infected with virus, with or without human THBP cDNA (pHTBr), and then collected by centrifugation (8). Expression of THBP was measured in a cell free preparation as previously described using ‘xl-labeled thyroid hormone binding protein complex (8). To study GSBP activity, 200 ~1 of packed cells (approximately 8 x lo6 cells) were suspended in a final volume of 1.0 ml of 50 mM Tris-HCl, pH 7.5, to which Triton X100 was added to a final concentration of 0.1% (v/v). The cells were sonicated by a Branson cell disruptor 185 at a number 5 setting for 5 seconds, followed by incubation on ice for 20 seconds The sonication and chilling steps were repeated 6 times. The resulting suspension was centrifuged in a Beckman ultracentrifuge Ty65 rotor at 233,000 x g for 1 hr. at 4°C. The supernate and resuspended pellet fractions were photolabeled as described below. Alternatively, 500 ~1 of packed cells (approximately 2 x lo7 cells, 9.5 mg of total protein) were suspended in a final volume of 1.0 ml containing 50 mM Tris-HCl, pH 7.5, 154 mM sucrose, 25 mM NaCl, 1 mM EDTA, 10 mM MnCl,, 10 mM ,+mercaptoethanol (TSNE buffer) with the following protease inhibitors: leupeptin (1.0 pg/ml), antipain (2.0 pg/ml), benzamidine (10.0 pg/ml) and aprotinin (1.0 pg/ml). The cells were sonicated under the conditions described above, but for 3 second intervals followed by incubation on ice for 30 seconds. Sonication and chilling steps were repeated 5 times. The suspension was centrifuged in a Beckman Microfuge 11 at 14,000 x g for 15 min. at 4°C. After the supernate was removed, the pellet was resuspended in an equal volume of buffer by brief (l-2 seconds) sonication. A clone containing the cDNA of human THBP was isolated from a human placenta $tll cDNA library (Clontech, Palo Alto, CA) using as probe a previously described (11) P-labeled 0.3 kbp EcoRI-Pvull fragment of bovine THBP cDNA. The resultant cloned DNA was subcloned in a plasmid pUC19. Then this 1.96 kbp DNA fragment (pT,BP), containing the coding region of the THBP lacking the coding region for signal peptide, 1320
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
was subcloned into the EcoRI-PstI site of plasmid pTrp781, which contained the a promoter, tetracycline resistance gene and plasmid replicon origin (12). E. coli MM294 was transformed by this recombinant plasmid. Transformed E. coli MM294 with or without the plasmid (pT,BP) was grown in 2xTy media (16 g/l Bacto tryptone, 10 g/l Bacto yeast extract, 5 g/l NaCl) to an absorbance of 0.4 at 550 nm. Two ml of the culture were transferred to 50 ml of minimum media (6 g/l Na,HPO,, 3 g/l IQ&PO,, 0.5 g/l NaCl, 1 g/l NH&l, 1% (w/v) glucose, 0.2% Casamino acids) containing tetracycline (12.5 pg/ml) and grown to the same absorbance. Expression of THBP was induced by addition of 3-indoleacrylic acid at a final concentration 25 pg/ml. After cultivation for an additional 4 hours, the cells were harvested by centrifugation at 3,000 rpm for 20 min. at 4”C, washed with 1.50 mM NaCl, then suspended in 1 ml of 10 mM Tris-HCl, pH 8.0 containing 10% sucrose, 200 mM NaCl, 1 mM EDTA and protease inhibitors. The cells were ruptured by sonication as described above for 10 seconds and incubated on ice for 30 seconds. The sonication and chilling procedure was repeated 5 times. Photolabeling of proteins with the ‘?-labeled azidopeptide was carried out as previously described (10). Supernate or membrane proteins from 3T3 cells or from E. &i in a final volume of 100 ~1 were incubated with 1 nmole of azidopeptide in the dark for 20 min. then irradiated at 254 nm for 2 min. After addition of 100 ~1 of SDS sample buffer, the sample was heated at 100°C for 2 min. and then subjected to SDS/PAGE followed by autoradiography. Labeling of the 57 kDa protein was quantitated using a densitometer. To measure the level of the lumenal marker P-glucuronidase in preparations of control and transformed 3T3 cell extracts, hydrolysis of 4-methylumbelliferyl glucuronide (UG) was determined. UG (5.0 mM) in 100 mM sodium citrate, pH 5.2, was incubated with the appropriate cell fraction at 37°C for 10 or 20 min. After termination of the reaction by addition of 0.1 N NaOH, fluorescence was measured by using a Perkin-Elmer LS3B fluorescence spectrometer at an excitation wavelength of 365 nm and an emission wavelength of 460 nm. RESULTS The discovery that hen oviduct GSBP had high sequence similarity to PDI, B-PH and THBP in various species (3) coupled with the finding that PDI, 8-PH and THBP were identical
(5,6,13), led us to consider if GSBP also was the same polypeptide.
The
approach taken was to carry out determinations of GSBP activity in both eukaryote and prokaryote cells expressing the THBP gene product. Our initial efforts focused on NIH 3113 cells transformed with human THBP cDNA. As previously reported (S), we found that the transformed cells contained a 55-57 kDa protein that bound ‘251-labeled T, and could be precipitated with a polyclonal antibody. Consistent with the results of earlier findings (8), the level of THBP was found to be approximately 4.5 times greater in cells transformed with the THBP cDNA than it was in control cells (Fig. 1A). Thus, introduction of the exogenous THBP cDNA markedly increased the level of THBP over the endogenous level found in the control cells. As expected if the THBP cDNA encodes for GSBP, when the ability to bind the glycosylation site photoprobe was assessed an approximately 5.5 fold increase in GSBP was detected (Fig. 1B). In a second experiment carried out in the absence of detergent, it was established that in ruptured 3T3 cells the 57 kDa labeled protein was localized to the 1321
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
,
RESEARCH
INSERT+ ’
SUP. --==mm
MEM.
COMMUNICATIONS
. kz’ 2%
INSERTSUP.
I MEM.
Figure
1. Expression of THBP and GSBP Activity in Transformed 3T3 Cells. A. Control cells (&ane 1) and transformedcells(Lane 2) were cooled to 15°Cand incubated with 0.5 nM ’ I-BrAc T3 for 1 hr. Cells were washed,extracted with 3 mM CHAPS and immunoprecipitatedwith polyclonal antibody 3632. The pellet was analyzed for labeled proteins after SDS/PAGE. El. Packedcells (200 ~1)were extracted with 0.1% Triton X100, sonicationand ultracentrifugation at 233,000x g for 1 hr. Supernatewas incubated with 1.0 nmole ‘251-azidopeptide probe at room temperaturefor 20 min., photolysedand electrophoresedon 7.5%-SDS/PAGE. In both A and B, ?labeled proteins were detected by autoradiographyand the relative level was measuredby densitometty. Figure 2. Distribution of GSBP Activity in Membrane and Supernatant Fractions of Transformed and Control Cells. Transformedcells (Lanes 1-6) or control cells (Lanes
8-13) were sonicatedin TSNE buffer and centrifugedat 14,000x g for 15 min. After the membranepellet was suspendedin the buffer, both the supernateand the membrane fractions were incubated with 1.0 nmole ‘=I-azidopeptide probe for 20 min. at room temperature, and photolysed. Both fractions were then subjectedto 7.5%-SDS/PAGE followed by autoradiography. The profile obtained with rat liver microsomeis shownin Lane 7.
membrane fraction, rather than the supernate fraction (Fig. 2) and that its level was markedly increased in the transformed cells when compared to the control celIs. Indeed, densitometry measurements, shown in Fig. 3, revealed that there was a linear response in
6Ins-
04 Firmre 3. Quantitation of the Level of %photolabeled Proteins in Transformed and Control 3T3 Cells. The relative levels of photolabeled proteins were quantified by
scanningof the autoradiogramsshownin Fig. 2. The level of the ‘ZI-photolabeledprotein in transformedcells (0) was 4.5-fold higher than that in control cells (0). Figure 4.
p-Glucuronidase
Activity in Microsomes
From Transformed
and Control
313
Cells. Cellswere sonicatedin buffer without Triton X-100, and then incubatedwith 5.0 mM UG in 100mM citrate buffer, pH 5.2 at 37°C. The reactionwasstoppedby addition of 0.1 N NaOH. Fluorescenceof UG from transformedcells (A) and control cells (A) at 10min. incubation or transformedcells (0) and control cells (0) at 20 min. incubation was measuredby Perkin-Elmer IS-3B FluorescenceSpectrometer. I322
vol.
170.
No.
3, 1990
BIOCHEMICAL
g "i 2
AND
E coli INSERT+ L-2 0s
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E. coli ALONE '0 Oa
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7
Fipure 5. Expression of GSBP Activity in E. coli. The total protein fraction from E. coJ transformedwith expressionvector containingTHBP cDNA (Lanes2,3), expressionvector alone (Lanes 45) or E. coli alone (Lanes 6,7) were incubated with 1.0 mnole ‘=Iazidopeptideprobe for 20 min. at room temperature,photolysedand analyzed by 7.5% SDS/PAGE. Lane 1 showsphotolabeledrat liver microsomes.
labeling with the azidopeptide increase in photolabeling
in the range of 20-100 pg of protein, and that the overall
in the cells transformed was 4.5 fold, in excellent agreement with
the observed increase in the level of THBP. Because it seemed possible, albeit unlikely, that transformation of human THBP cDNA into 3T3 cells induced a general increase in the level of microsomal proteins, we determined
the level of the microsomal
marker
enzyme, @-glucuronidase,
in both
transformed and control 3T3 cells. As shown in Fig. 4, under conditions where microsomal enzyme activity was linearly dependent on the amount of protein and time of incubation
there was no difference in the level of this microsomal
control and transformed
marker
enyzme in
cells.
Finally, we examined E. coli cells that had been transformed with the human TJXBP cDNA.
As shown in Fig. 5, the THBP transformed E. coli expressed a 55-57 kDa protein
that binds the photoaffinity probe, whereas neither E. coli containing coli alone expressed this protein.
only vector nor E.
The results of these studies allow one to expand the list of properties of the human 57 kDa protein found in the lumen of the endoplasmic reticulum to include binding of a peptide containing a N-glycosylation site. Thus, it appears that a single 55-57 kDa lumenal protein functions: 1) in the form of a homodimer that functions to scramble disulfide bonds (PDI); 2) as the B component of the CY& tetramer that hydroxylates prolyl residues (B-PH);
3) as a binding protein that recognizes elements in the structure of thyroid hormone (THBP); and 4) as a binding protein that recognizes N-glycosylation site peptides (GSBP). The possibility that this apparently multifunctional binding protein plays a role in protein maturation in the lumen of the endoplasmic reticulum is intriguing. ACKNOWLEDGMENTS:
and NIH Postdoctoral
This work was supported by NIH Grant GM 33185 to W.J.L., Fellowship GM12628
to R.N.
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REFERENCES 1.
2. 3. 4.
Welply, J.K., Shenbagamurthi, P., Naider, F., Park, H.R., and Lennarz, W.J. (1985) J. Biol. Chem. 260, 6459-6465. Kaplan, H.A., Welply, J.K., and Lennarz, W.J. (1987) Biochim. Biophys. Acta 906, 161-173. Geetha-Habib, M., Noiva, R., Kaplan, H.A., and Lennarz, W.J. (1988) Cell 54, 1053-1060. Edman, J.C., Ellis, L., Blancher, R.W., Roth, R.A., and Rutter, W.J. (1985) Nature 317, 267-270.
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Cheng, S., Gong, Q., Parkinson, D., Robinson, E.A., Appella, E., Merlino, G.T., and Pastan, I. (1987) J. Biol. Chem. 262, 11221-11227. Pihlajaniemi, T., Helaakoski, T., Tasanen, K., Myllyla, R., Huhrala, M-L., Koivu, J., and Kivirikko, K.I. (1987) EMBO J. 6, 643-649. Parkkonen, T., Kivirikko, K.I., and Pihlajaniemi, T. (1988) Biochem. J. 256, 10051011. Kato, H., Velu, T., and Cheng, S-Y. (1989) Biochem. Biophys. Res. Commun. 164, 238-244. Kaplan, H.A., Naider, F., and Lennarz, W.J. (1988) J. Biol. Chem. 263, 7814-7820. Welply, J.K., Kaplan, H.A., Shenbagamurthi, P., Naider, F., and Lennarz, W.J. (1986) Arch. Biochem. Biophys. 246, 808-819. Yamauchi, K., Yamamoto, T., Hayashi, H., Koya, S., Takikawa, H., Toyoshima, T., and Horiuchi, R. (1987) Biochem. Biophys. Res. Commun. 146, 1485-1492. Kurosawa, T., Sasada, R., Iwane, M., and Igarashi, K. (1987) Nucl. Acid Res. 11, 3077-3085. Koivu, J., MyIlyia, R., Helaakoski, T., Pihlajaniemi, T., Tasanen, K., and Kivirikko, K.I. (1987) J. Biol. Chem. 262, 64476449.
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