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a Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
b Unité de recherche en pédiatrie, Centre hospitalier universitaire, Université Laval, Ste-Foy, Québec, Canada G1V 4G2
c Faculté de médecine vétérinaire, Université de Liège, Liège, Belgium
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We report herein the characterization of a granulosa cell 58-kDa protein following immunoscreening of bovine luteal and granulosa cell expression cDNA libraries. Characterization of these cDNAs revealed that they encode for a protein of 60.1 kDa with a signal peptide. Homology search in GenBank revealed that bovine 60.1-kDa protein is related to a human protein named 80K-H [5, 6] and to a mouse protein named putative ß subunit of glucosidase II (ß-GII) [7]. The biological role of the human 80K-H is unclear, as it was recently linked to intracellular trafficking associated with advanced glycation end product (AGE) receptors [8] and to intracellular signaling associated with the fibroblast growth factor III receptor [9, 10], and for the mouse counterpart to the process of protein maturation in the endoplasmic reticulum [7, 11]. We report herein that the bovine 60.1 kDa is an unglycosylated acidic membrane protein found associated with intracellular membranes. We have named this bovine 60.1-kDa protein vacuolar system-associated protein or VASAP-60.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chemical reagents used were obtained from Sigma Chemical Co. (St. Louis, MO) if not otherwise stated.
Partial Purification of the 58-kDa Protein (p58) and Polyclonal Antibody Production
Follicles were obtained from cycling cows killed at a local slaughterhouse. Ovaries were immediately transported to the laboratory in PBS (0.6 mM KH2PO4, 4.4 mM Na2HPO4, 0.15 M NaCl, pH 7.4) on ice. Granulosa cells were recovered after follicular puncture and washed in PBS by four centrifugations (500 x g, 10 min, 4°C) to remove follicular fluid, red blood cells, and cellular debris. Cells were frozen, lyophilized, resuspended in water (30°C), and centrifuged (50 000 x g, 1 h, 4°C) to collect the supernatant soluble proteins. Granulosa cell protein extracts were precipitated using 25% (NH4)2SO4 saturation (2–4 h at 4°C) and then centrifuged (7000 x g, 1 h, 4°C). The supernatant was chromatographed on a phenyl Sepharose CL4B column (Amersham Pharmacia Biotech, Pointe-Claire, PQ, Canada) previously equilibrated with glycine buffer (20 mM, pH 9) with 25% (NH4)2SO4. The column was developed in four steps with glycine buffer of decreasing ionic strength (25%, 12%, 0% [NH4]2SO4) and finally with 50% ethylene glycol (v:v). The four eluted fractions were extensively dialyzed against 5 mM NH4HCO3, pH 8, and then lyophilized prior to SDS-PAGE analysis. Each fraction was subjected to one-dimensional SDS-PAGE on a 9–15% gradient slab gel in reducing buffer conditions as described previously [12]. After electrophoresis, the proteins were either visualized in the gel via Coomassie Blue R250 staining or transferred to a nitrocellulose membrane according to Towbin et al. [13], which was then stained using Ponceau S Red. The p58 band was cut, dissolved in 500 µl of dimethyl sulfoxide, and mixed with an equal volume of Freund's complete adjuvant before subcutaneous injections in rabbits (80 µg/immunization) as described by Knudsen [14]. Booster injections were given at 2-wk intervals for 3 mo; then rabbits were alternatively bled or immunized every 2 wk for 6 mo. Serum was collected following overnight coagulation (4°C), aliquoted, and stored frozen (-20°C) until used in further immunoblotting or immunoprecipitation analysis.
One-Dimensional SDS-PAGE and Immunoblotting of VASAP-60
Total protein extracts from different bovine tissues or from different species were obtained after homogenization in ice-cold Tris buffer (60 mM Tris-HCl, 5 mM EDTA, pH 6.8) in the presence of protease inhibitors (1 mM PMSF, 2.5 µg/ml leupeptin, 2.5 µg/ml aprotinin, 1.2 µg/ml pepstatin-A, 2 mM benzamidine). After centrifugation (28 000 x g, 20 min, 4°C), the supernatant was recovered. Protein concentration was determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA) with BSA as standard (fraction V; Sigma). Protein extracts (100 µg/well) were size fractionated on a one-dimensional discontinuous polyacrylamide-SDS slab gel in reducing conditions after incubation at 100°C for 5 min [12]. After electrophoresis, the proteins were either visualized via Coomassie Blue staining or transferred overnight at 4°C to nitrocellulose membrane (0.45-µm Hybond-C; Amersham) and stained with Ponceau S Red. Immunological detection was performed according to Harlow and Lane [15]. Briefly, nonspecific binding sites were blocked by incubation of the nitrocellulose membrane in a TNT buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% Tween 20) with 20% normal calf serum (NCS). The membrane was then incubated with the rabbit polyclonal anti-p58 antibody diluted at 1:1000 in a solution of TNT with 10% NCS. Antigen and first antibody complexes were revealed after incubation with a second antibody (goat anti-rabbit IgG linked with horseradish peroxidase antibody [H & L chains; human serum adsorbed; Life Technologies, Burlington, ON, Canada]) in TNT buffer with 10% NCS and developed with 4-chloro-1-naphtol (CN; Gibco-BRL, Gaithersburg, MD) in TN buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5).
Establishment of the cDNA Expression Library
Bovine corpus luteum and granulosa cell cDNA expression libraries were established to allow characterization of the granulosa-luteal cell p58. Bovine corpora lutea from Day 5 to Day 10 of the estrous cycle were obtained at a local slaughterhouse, immediately dissected from the ovarian stroma, and frozen in liquid nitrogen. Bovine ovaries from Day 3 of superovulation (eCG, 3000 IU; Ayerst, Montreal, PQ, Canada) were obtained, and granulosa cells were aspirated and frozen in liquid nitrogen. Corpus luteum and granulosa cells were homogenized in guanidium buffer (4 M guanidium isothiocyanate, 0.5% sodium N-laurylsarco-sine, and 25 mM sodium citrate, pH 7; [16]) and then centrifuged (192 000 x g, 20 h, 20°C) on a cesium trifluoroacetic acid cushion (Amersham). Messenger RNA was isolated with oligo-(dT)25 magnetic beads (Dynabeads; Dynal, Lake Success, NY) once and then reversed transcribed with Superscript II (Superscript Choice System for cDNA synthesis; Gibco-BRL) and oligo-(dT)12 as described previously [17]. The cDNA were ligated with EcoRI (NotI-SalI) adaptors, size fractionated on a 1-ml Sephacryl S-500 HR column according to the manufacturer's recommendations (Superscript Choice System for cDNA synthesis; Life Technologies, Gaithersburg, MD), and then ligated to lambda Zap Express EcoRI/CIAP phage arms (Stratagene, La Jolla, CA) followed by in vitro packaging (Gigapack II packaging extract; Stratagene).
Immunological Screening of the cDNA Expression Libraries
The bovine corpus luteum and granulosa cell phage libraries were used to infect Escherichia coli XL1-Blue MRF' (Stratagene), plated at a density of 50 000 plaque-forming units (pfu) on 150-mm NZY 0.7% top agarose plates [18], and incubated at 42°C for 3.5 h. Nitrocellulose membranes (Hybond-C, 0.45 µm; Amersham) were soaked in 10 mM isopropyl ß-D-thiogalactopyranoside (IPTG) and then overlaid on NZY top agarose and incubated at 37°C for 10 h. The nitrocellulose membranes were then treated as described for immunoblotting. To screen the cDNA expression libraries, the polyclonal anti-p58 and the anti-VASAP-60-C antibodies were preincubated with 1 mg/ml of E. coli total protein extract for 30 min preceding incubation with the nitrocellulose membrane. A first round of screening of the corpus luteum library was accomplished with the anti-p58 polyclonal antibody. Four clones were purified and characterized by sequencing. Subsequently, a second round of screening was achieved with the anti-VASAP-60-C antibody (see section on expression of recombinant VASAP-60) on the granulosa cell cDNA expression library. Thirteen clones were purified and characterized by sequencing. Phagemid BlueScript pBK-cytomegalovirus was in vivo excised from lambda Zap Express-positive clones. Recombinant phagemids were isolated by alkaline lysis [18], purified via Magic Mini prep columns (Promega, Madison, WI), and then digested with EcoRI. Products of digestion were size fractionated by electrophoresis on a 1% agarose gel and visualized under UV illumination using ethidium bromide. Positive recombinant phagemid clones were sequenced by the double-stranded dideoxychain termination method using the T7 polymerase (T7 Sequencing Kit; Amersham) with T3 and T7 oligonucleotide primers or by cDNA-specific oligonucleotide primers. The consensus nucleotide sequence of two independent clones (double-stranded sequenced) is reported. Amino acid sequence was deduced from the nucleotide sequence and analyzed for hydrophobicity, isoelectric point, secondary structure, and conserved patterns using the data bank PROSITE [19] and programs provided by Genetics Computer Group software (ver. 8, 1994; Madison, WI). Amino terminus signal sequence and prediction of protein cellular localization sites were analyzed by the program PSORT [20].
Expression of Recombinant VASAP-60 and Production of Antisera
The original cDNA sequence of the VASAP-60 was modified to remove the 5' noncoding region from the original cDNA. A sense oligo incorporating a BamHI restriction site (5'-GATGGATCCATGTTACTGCTGCTGCTGCTACTG-3') was designed to allow amplification from the ATG codon of VASAP-60, and this was paired with an anti-sense oligo (5'-GCCATCCATGTCATCATCC-3') downstream of an endogenous HincII restriction site located at 274 base pairs (bp) of the VASAP-60 ATG codon. Polymerase chain reaction (PCR) amplification was performed using the enzyme Expand High Fidelity (Roche Molecular Biochemicals, Laval, PQ, Canada) as specified by the manufacturer's protocol. A 678-bp PCR product was generated that was then cloned in pGem-T (Promega). The original VASAP-60 cDNA was digested by HincII and EcoRI and ligated with the 678-bp PCR fragment into pGEM-T. The complete VASAP-60 cDNA was then digested by BamHI and EcoRI and subcloned into pGEX-2T (Amersham) in frame with the glutathione-S-transferase (GST) protein. A second VASAP-60 construction representing the strongest hydrophilic portion of VASAP-60 from 246A to 418S was generated by PCR amplification using the enzyme Expand High Fidelity with a sense primer (5'-GATGGATCCGCATTATCAGAAGGGGAAGCC-3') and an anti-sense primer (5'-TCAGAATTCCGCTGTACAGGTAGGCGAACTCG-3') that included engineered BamHI and EcoRI restriction sites, respectively. The 538-bp PCR product was then cloned into pGEX-2T in frame with the GST protein. The first construct was designed to generate a complete recombinant VASAP-60 protein of 533 amino acids (60.1 kDa, isoelectric point [pI] 4.2), whereas the second construction, named VASAP-60-C, was designed to generate a 173-amino acid protein (18.4 kDa, pI 3.6) of the central domain of the protein. Both plasmid constructions, the complete VASAP-60-GST and the VASAP-60-C-GST, were verified by sequencing for their in-frame position within GST and for potential mutations following PCR. Both clones were transformed into a protease-deficient E. coli strain (BL-21; Amersham), and protein expression was induced by IPTG for 4 h when optical density reached a value of 0.5 at 600 nm. As recommended in the manufacturer's protocol (Amersham), bacterial protein extracts were obtained after sonication and centrifugation, generating soluble and nonsoluble fractions (including inclusion bodies). Protein extracts from both constructions were analyzed on a denaturing 7.5% SDS-PAGE gel. Only the VASAP-60-C-GST was expressed in both fractions when the gel was stained with Coomassie Blue. The complete VASAP-60-GST was present only in low concentration in the nonsoluble fraction as revealed by Western blotting. The VASAP-60-C-GST hybrid protein was purified on glutathione Sepharose beads (Amersham), digested with thrombin to release the recombinant VASAP-60-C from GST, purified on a 12% denaturing SDS-PAGE gel, transferred onto nitrocellulose, and stained with Ponceau S Red. The protein band corresponding to the VASAP-60-C was cut and processed to produce polyclonal antibodies as described in the first section of Materials and Methods.
Northern Analysis of VASAP-60
Transcriptional analysis was performed with different bovine tissues collected at a slaughterhouse. Tissues (2 g) were homogenized with a Polytron (8000 rpm; Kinematica AG, Luzern, Switzerland) in 20 ml of guanidium buffer followed by two cycles of extraction and precipitation in phenol-chloroform and isopropanol [16]. RNA molecular weight markers from 9 kilobases (kb) to 0.25 kb (Life Technologies) were used to estimate sizes of transcripts. Total RNA (20 µg) was size fractionated on a 0.66 M formaldehyde, 1% agarose gel and transferred overnight by capillarity (10-strength SSPE; [18]) to a nylon membrane (Hybond-N; Amersham) and UV treated (150 mJ). The amount of rRNA (28S and 18S) was estimated after methylene blue staining [21]; the image was digitized (FotoDyne Inc., Hartland, WI) and analyzed using the software Collage (FotoDyne). The full 2.4-kb cDNA probe was used to generate radioactive probes where [32P]dCTP (NEN Life Sciences, Boston, MA) was incorporated by the random priming method (T7 Quickprime; Amersham). The blotted membranes were prehybridized at 65°C in a rotating hybridization oven with 4-strength SET (0.6 M NaCl, 0.12 M Tris, 7.4, 4 mM EDTA), 0.1% Na4P2O7, 0.2% SDS, and 500 µg/ml heparin for 4 h; they were then hybridized at 65°C overnight with the radioactive probe including 10% dextran sulfate [22]. Membranes were then washed with double-strength SSPE at room temperature for 15 min, further washed with 0.1-strength SSPE + 0.1% SDS at room temperature for 30 min, and finally washed under stringent conditions at 65°C for 1 h in the same buffer. The membranes were exposed for one day to photographic film (Biomax-MR; Eastman Kodak, Rochester, NY) at -70°C with an intensifying screen. The film images were digitized as described above, and the intensities of bands were expressed as ratios of 28S rRNA to account for procedural losses.
Luteal Cell Culture
Luteal cells were obtained from bovine corpora lutea of Day 5 to Day 10 of the estrous cycle that was collected from a slaughterhouse and transported (45 min) on ice in sterile PBS. Luteal cells were dissociated according to Simmons et al. [23] with modifications. Pieces (1–2 mm3) of luteal tissue were digested in a spinner flask at 37°C in RPMI (Life Technologies), 25 mM glucose, 0.1% ovalbumin, 2 mM glutamine, 20 mM Hepes, 50 U/ml collagenase (Life Technologies), 2.4 U/ml dispase (Life Technologies), and 200 U/ml DNase. The luteal cells from the first digestion (45 min) were discarded; then luteal cells from subsequent digestions of 45 min were collected, washed twice in RPMI, pooled, and kept on ice. Mean viability for multiple cell culture experiments was estimated at 91% by the trypan blue exclusion method. Cells were cultured overnight to allow for plating in RPMI supplemented with 2 mM glutamine, 20 mM Hepes, 40 IU penicillin, 40 µg streptomycin, and 0.1 µg Amphotericin B (Life Technologies), and 10% NewBorn fetal calf serum (FCS; Life Technologies) and plated at a density of 10 x 106 cells/75 cm2.
Subcellular Fractionation
Luteal cell nuclei were isolated from luteal cells in culture by centrifugation on a sucrose gradient according to Greenberg and Bender [24]. Before isolation of nuclei, luteal cells were washed in ice-cold PBS several times to remove cellular debris and red blood cells. The cells were dissociated from culture dishes on ice by treatment with 0.05% trypsin, 0.5 mM EDTA in Dulbecco's modified Eagle's medium without Ca2+ and Mg2+ (Life Technologies). Membrane fractions were generated by homogenization on ice of corpora lutea in 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 2 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF, 2.5 µg/ml leupeptin, 2.5 µg/ml aprotinin, 1.2 µg/ml pepstatin-A, and 2 mM benzamidine. The homogenates were centrifuged (28 000 x g, 20 min, 4°C), and the supernatants were recovered and centrifuged (35 000 x g, 20 min, 4°C). The pellets were resuspended in homogenization buffer; this constituted the membrane fraction. The supernatant from the latter fraction was centrifuged (120 000 x g, 1 h, 4°C), and the recovered supernatant constituted the cytosol fraction. The cytosol fraction was lyophilized and reconstituted in buffer. Protein concentrations for each fraction were quantified by the Bradford assay. To localize VASAP-60, proteins from subcellular fractions (100 µg) were subjected to denaturing 10% SDS-PAGE and transferred onto a nitrocellulose membrane for immunoblotting with the anti-recombinant VASAP-60-C antibody. Glycosylation analysis of VASAP-60 was achieved followed by overnight endoglycosidase H (Roche Molecular Biochemicals) digestion of bovine membrane corpus luteum extracts. The endoglycosidase H digestion protocol was carried out as described by Freeze [25]. After overnight treatment with endoglycosidase H, protein extracts were analyzed by Western blotting using the anti-recombinant VASAP-60-C.
The Golgi/endosome (G/E) fraction was prepared as described previously [26]; it had been characterized earlier with respect to marker enzymes, cytochemistry, electron microscopy, and ligand-mediated endocytosis and shown to contain Golgi elements but to be substantially free of plasma membrane and other subcellular constituents [27]. Briefly, the rat liver protein extracts were obtained after homogenization in 4 ml of buffer (0.25 M sucrose, 25 mM KCl, 50 mM Hepes, 1 mM PMSF, 2 mM sodium orthovanadate, 1 mM benzamidine, pH 7.4) per gram of liver in a Potter Elvehjem homogenizer with six passes of a motorized polyethylfluoroethylene pestle at 1500 rpm. The homogenates were centrifuged at 3000 x g for 10 min. The supernatant was then centrifuged at 200 000 x g for 40 min, and the pellet was resuspended and adjusted by using a refractometer to a final concentration of 1.15 M sucrose. This was placed below 1 M and 0.6 M sucrose cushions. After centrifugation at 96 000 x g for 205 min, the endosomes at the 0.6–1.0 M interface were collected and used immediately. For purification of glycoproteins, endosomes (5 mg cell fraction protein) were solubilized by agitation at 4°C for 30 min in the presence of 1% Triton X-100. Solubilized membranes were centrifuged to remove undissolved materials, and the supernatant was applied to WGA-Sepharose (2-ml packed columns, 5 cycles at 4°C; Amersham). The columns were washed with 40 column volumes of 50 mM Hepes (pH 7.6) containing 1 mM PMSF, 1 mM benzamidine, 1 mM vanadate, 40 mM sodium fluoride, and 0.1% Triton X-100 and centrifuged at 500 x g for 2 min. Glycoproteins were eluted by resuspension of each column in 2 ml of 0.3 M N-acetyl-D-glucosamine for 60 min at 4°C. The columns were spun to yield the eluates (60–100 mg/ml protein). Protein content in the fractions was determined by a modification of Bradford's method using BSA as standard [28].
Immunohistochemistry
Immunohistochemistry was performed on PBS-buffered formaline-fixed bovine tissues collected at the slaughterhouse. Paraffin-embedded tissues were cut at 8-µm thickness, mounted on poly-L-lysine slides, deparaffined, and rehydrated. Tissue sections were treated as previously described [29] and incubated for 16 h at 4°C with the rabbit anti-recombinant VASAP-60-C antibody at a dilution of 1:500 in Tris-buffered saline (TBS) including 1% normal cow serum. Negative control tissue sections were incubated similarly with a nonimmune rabbit serum. The first antibody bound to VASAP-60 was revealed with a mouse monoclonal anti-rabbit IgG conjugated to alkaline phosphatase at a dilution of 1:100 for 2 h at room temperature, followed by several washes in TBS. Visualization was performed by the alkaline phosphatase reaction with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche Molecular Biochemicals). Photographs were taken under brightfield illumination using a Zeiss (Carl Zeiss, Thornwood, NY) microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The bovine granulosa cell p58 protein was eluted predominantly from the phenyl Sepharose chromatography in the third chromatographic step gradient with glycine buffer (20 mM, pH 9) without (NH4)2SO4, as observed on the 9–15% gradient SDS-PAGE gel (Fig. 1A). After electrophoresis and transfer onto nitrocellulose, the protein band corresponding to p58 was cut and used to raise polyclonal antibodies in rabbits. These antibodies were shown to bind specifically to a protein of 58–60 kDa in total protein extracts of corpus luteum, testis, liver (Fig. 1C), and granulosa cells (data not shown). Characterization of the granulosa cell p58 was achieved by screening a 
ZAP bovine cDNA corpus luteum expression library. After the first immuno-screening of 3 x 105 pfu with the anti-p58 antibody, four positive clones were purified to homogeneity, followed by phagemid in vivo excision and further characterization. Two cDNA of approximately 1.9 kb and two of 2.2 kb were obtained. Sequencing of the four clones revealed the same nucleotide sequence except for a small 5' region present in the 2.2-kb clones but absent in the 1.9-kb clones.
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Complementary DNA Sequence Analysis of VASAP-60
Complete sequencing of the two 2.2-kb clones by double-stranded sequencing revealed a cDNA of 2095 bp. Analysis of the sequence revealed a 5'-untranslated (UTR) region composed of 141 bp, an open reading frame (ORF) of 1599 bp, and a 3'-UTR of 355 bp that included two polyadenylation signals followed by a poly(A)+ tail. The longest ORF, using the first in-frame ATG, coded for a protein of 533 amino acids of an estimated molecular mass of 60.1 kDa and an isoelectric point of 4.2 (Fig. 2). Hydropathy profile analysis based on the method of Kyte and Doolittle [30] with a window frame of 20 residues showed a hydrophilic protein with a short hydrophobic region at the amino terminal end composed of 16 amino acids displaying a potential alpha helix secondary structure. Analysis of VASAP-60 amino acid sequence by PSORT software revealed a potential leading signal peptide of 13 amino acids. Removal of this 13-amino acid leading peptide signal would yield a mature protein of an estimated molecular mass of 58.6 kDa.
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An amino acid homology search in GenBank using the BLAST search program revealed an overall 86.7% identity to a human protein when aligned for maximum matching homology. This human protein had an estimated molecular mass of 59.2 kDa and was named 80K-H (Fig. 3). In addition, a recently cloned murine protein of 58.7 kDa, named putative ß-GII, showed an overall identity of 81.8%. Differences in amino acid sequence between the bovine VASAP-60, the human 80K-H, and the murine putative ß-GII were more limited in specific segments. An 86-amino acid segment (278K to 363P) located in the middle portion of VASAP-60 was only 63.9% identical to the 80K-H and 52.3% or 59.3% to the ß-GII or its isoform ß-GII-B1, respectively. This amino acid segment corresponded to the strongest hydrophilic region of the protein and was composed of an acidic domain (AC) containing glutamic and aspartic amino acids positioned in tandem (313E-334D). Interestingly, this acidic domain was flanked upstream and downstream by proline-rich amino acid stretches—288YPPSPPAP, 301KEEQPPMP, and 341KDAPPPAP—which may accommodate cognate SH3 domains (Dr. Lewis Cantley, Harvard Medical School, personal communication). The amino acid region found between 116K and 211E contained 53% potentially charged amino acids (H, K, R, D, E) included in potential hydrophilic alpha helices (Figs. 2 and 3). Analysis of the amino acid potential secondary structure showed that the amino and carboxyl terminal ends of VASAP-60 may adopt a globular structure separated by multiple hydrophilic alpha helices. Within these predicted amino and carboxyl globular domains were found clustered all 17 cysteines that are conserved in comparison to the human 80K-H and mouse ß-GII (Fig. 3).
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Amino acid consensus motif analysis was performed on the VASAP-60 using the data bank PROSITE. Two potential N-linked glycosylation sites were located at 71NGSF and 481NRST. Multiple potential protein kinase C and casein kinase II phosphorylation sites were found on threonine and serine residues. The sequences 221DDDMDGAVSVAE and 240DTDGDGALSEGE represent potential EF-hand calcium-binding domains, whereas amino acid sequence 530HDEL, found at the carboxyl terminal end, represents a potential endoplasmic reticulum (ER) targeting sequence.
Procaryotic Expression of Recombinant VASAP-60
Recombinant VASAP-60 (total) or VASAP-60-C (central fragment) was produced in bacteria as hybrid proteins with GST to show that the original anti-p58 antibody recognizes protein expressed by the cloned VASAP-60 cDNA, and to raise a polyclonal antiserum against a defined segment of the recombinantly produced VASAP-60. The total VASAP-60-GST was expressed at a low level and found only in the nonsoluble fraction (inclusion bodies) as detected as a faint band by Western blotting analysis using the original anti-p58 antibodies (data not shown). The VASAP-60-C-GST protein was found equally in the soluble and nonsoluble fractions as a 54-kDa protein band when analyzed by Coomassie Blue staining or Western blotting using the original anti-p58 antibody (Fig. 4A). The VASAP-60-C-GST was purified by affinity on agarose glutathione beads and was digested by thrombin to release the VASAP-60-C fragment. The purified VASAP-60-C was then analyzed by SDS-PAGE, electrotransferred onto nitrocellulose, and stained by Ponceau S Red. The VASAP-60-C migrated at around 43 kDa instead of 18.4 kDa as estimated from the cDNA (data not shown). In Western blotting analysis, the original anti-p58 antibody recognized the recombinant VASAP-60-C at 43 kDa (Fig. 4B). We have raised polyclonal antibodies against the purified recombinant VASAP-60-C. The antibodies were used to analyze cross-reacting proteins in corpus luteum (Fig. 4C). The anti-VASAP-60-C antibodies recognized three unglycosylated protein bands in Western blotting of corpus luteum total protein extracts that were treated or not treated with endoglycosidase H. A major 88-kDa and two minor 58-kDa and 50-kDa proteins were detected (Fig. 4C and 6). The anti-recombinant VASAP-60-C antibodies were then used to screen the bovine granulosa cell ZAP expression cDNA library in hopes of characterizing other proteins related to VASAP-60. Thirteen positive clones were purified and showed cDNA insert size of around 2 kb. The sequencing of the 13 clones revealed nucleotide sequences identical to the original VASAP-60 sequence as shown in Figure 2.
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Expression Studies of VASAP-60
Expression analysis of VASAP-60 was performed via Northern blot on total RNA isolated from various bovine tissues to search for its pattern of expression. A single transcript corresponding to 2.3 kb was observed in all tissues studied (Fig. 5). Analysis of steady state VASAP-60 mRNA levels expressed as ratios of 28S rRNA indicated that levels of expression varied between tissues; they were higher in adult testis, liver, eCG-treated follicular wall, and stomach and lowest in the heart (atrium or ventricle). Total protein extracts of various bovine tissues, analyzed by Western blotting using the anti-VASAP-60-C antibody, showed the presence of three protein bands of 88 kDa, 58 kDa, and 50 kDa (Fig. 6A). The concentration of these three proteins varied between tissues. The 88 kDa was the most conspicuous protein, found in most tissues studied except for the duodenum, whereas the 58 kDa and 50 kDa were detected at lower levels and were found in all tissues. The level of conservation of VASAP-60 expression in various species was analyzed in Western blotting of total liver protein extracts using the anti-VASAP-60-C antibody. The 88-kDa protein band was well detected in the bovine and human species. The 58-kDa and 50-kDa proteins were observed only in the bovine species. No cross-reacting protein was observed in the dog, horse, rat, mouse, chicken, fish (Fig. 6B), amphibian, reptile, Caenorhabditis elegans, or Drosophila melanogaster (data not shown). The interspecies cross-reaction studies using anti-VASAP-60-C antibody agreed with the fact that these antibodies were raised against the least conserved fragment of VASAP-60, 246A-418S, in comparison to human and murine amino acid (Fig. 3).
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Cellular fractionation methods were used to assess the cellular compartmentalization of VASAP-60. We observed that VASAP-60 was mainly localized in the membrane fraction of bovine corpus luteum in comparison to the cytosol fraction when the anti-VASAP-60-C antibody was used. In the membrane fraction, the 88-kDa protein was the strongest in comparison to the 58-kDa and 50-kDa protein bands. These latter two bands were undetectable in the cytosol fraction (Fig. 6C). No signal was observed in the nuclear fraction. We used a highly purified G/E fraction from the rat liver to further assess the intracellular compartmentalization of VASAP-60. The rat liver was used because the methodology was validated for this model, in combination with use of the original anti-p58 antibody since the anti-VASAP-60-C antibody does not recognize the rat protein. A signal for p58 was easily detected both in the whole endosomal (G/E) fractions and in WGA chromatographic eluates of the G/E fraction. These results support the premise that VASAP-60 is present in internal membrane and may be associated with glycoproteins enriched by WGA affinity chromatography, since we have shown that VASAP-60 is not glycosylated following treatment with endoglycosidase H.
Immunohistochemical studies showed a specific pattern of VASAP-60 expression in bovine female reproductive tissues. In the ovary, VASAP-60 was localized in the oocyte, cumulus and granulosa cells, and theca cells of follicles (Fig. 7, A and B). Oocytes from primary to preovulatory follicles were stained (Fig. 7B). VASAP-60 was not expressed in ovarian stromal tissue. VASAP-60 was also observed in the uterus in association with the endometrial epithelial and glandular cells (Fig. 7C) but not in endometrial stromal tissue. Renal tubular epithelial cells showed strong labeling associated with intracellular vesicles, whereas nuclei were not stained (Fig. 7D). VASAP-60 labeling was associated with oviductal epithelial cells whereas their nuclei and stroma were not stained (Fig. 7, E and F). Positively immunostained epithelial cells showed VASAP-60 localization predominantly in their perinuclear region, and in their pericytoplasmic membrane region as observed in the oocyte (Fig. 7B) and in renal epithelial tubular cells (Fig. 7D). Furthermore, VASAP-60 immunolabeling indicated a basolateral and/or apical polarity as observed in the endometrial glandular cells (Fig. 7C) and oviductal epithelial cells (Fig. 7, E and F). The staining was clearly associated with intracellular vacuoles or vesicles in oocytes, epithelial oviductal cells, and renal tubular epithelial cells.
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DISCUSSION |
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Homology searches in GenBank revealed an overall 86.7% and 81.8% identity when amino acids were aligned for maximum matching homology with a human protein termed 80K-H [5] and a mouse protein termed putative ß-GII [7]. The human 80K-H was purified in parallel with the 80K-L by Hirai and Shimizu [6] while searching for protein kinase C (PKC) substrate proteins. Both proteins migrate at around 80 kDa with a small difference in their electrophoretic mobility and were thus named the 80K-L (for light) and 80K-H (for heavy) proteins. The 80K-L was shown to be the human MARCKS protein, a strong PKC substrate, whereas 80K-H bore no relationship to MARCKS and was shown to be a poor in vitro PKC substrate [5, 6]. Recently, the 80K-H was shown to be involved in the formation of a membrane protein complex involved in the signal transduction of the fibroblast growth factor III receptor [9, 10]. Furthermore, the biological role of the 80K-H has been extended to cellular membrane trafficking following its interaction with AGE-specific receptors located at the cell surface and also in relation to intracellular compartments [8]. Glucosidase II (GII) is a known resident ER protein involved in the maturation of N-linked oligosaccharide proteins [11, 33, 34]. However, it is still a question for debate whether GII is composed solely of a single polypeptide chain [33, 35] or whether it adopts a quaternary structure and is found as a heterodimer composed of a ~110-kDa catalytic 
subunit and an 80-kDa or 58-kDa noncatalytic ß subunit [7, 11, 34].
VASAP-60 is 6, 12, or 4 amino acids longer than the human 80K-H, the murine putative ß-GII, or its recently cloned isoform, ß-GII-B1 proteins [36], respectively. These amino acid insertions are centrally located within the most hydrophilic segment of the protein that is composed of acidic and proline-rich sequences; we have named this segment the SH3-acidic-SH3 putative ligand-binding domain (see below). The putative SH3-acidic-SH3 domain of VASAP-60 (278KYRS to RMPP363) shows only 63.9%, 52.3%, or 59.3% identity when compared to 80K-H, ß-GII, or ß-GII-B1 proteins, respectively. Our Western blotting data present a discordant electrophoretic mobility profile for VASAP-60 in comparison to the molecular weight deduced from its cDNA or when compared to published human, mouse, and rat proteins. The human 80K-H protein was detected around 80 kDa [5, 6] or 90 kDa [8–10], and the mouse and rat proteins were detected around 80 kDa [7, 11]. Of note, in the latter studies, the 80-kDa value for the rat protein was obtained using reducing SDS-PAGE, while in nonreduced conditions the protein migrated faster to a value of 58 kDa [11]. In contrast, our original anti-p58 antibody revealed a cross-reacting bovine, human, rat, and mouse protein near 58 kDa, whereas no protein band was observed in the 80-kDa to 90-kDa range for all species (data not shown). However, the use of the anti-recombinant VASAP-60-C antibodies in Western blotting of total protein extracts from various bovine tissues revealed a major 88-kDa and two minor 58-kDa and 50-kDa protein bands. Interestingly, it has been shown that the tyrosine-phosphorylated human 80K-H interacts with the SH2 domain of Grb2 [9]. In these GST pull-down experiments using a Grb2-(SH2)-GST fusion protein incubated with whole protein extracts, the presence of the 80K-H in its tyrosine-phosphorylated form was detected. However, when the same protein blot was stripped and reincubated with anti-peptide antibodies raised against amino acids corresponding to the first 14 amino and the last 14 carboxyl terminal ends of the signal peptide cleaved human 80K-H protein, the 80K-H protein band and a conspicuous 60-kDa protein band were revealed (Fig. 6 of Goh et al. [9]). The 14 amino acid peptides corresponding to the amino and carboxyl terminal ends used to generate antibodies against the human 80K-H show a high degree of homology when compared to VASAP-60. Thus, the near 60-kDa protein that Goh et al. [9] have identified in relation to the human 80K-H may be related to the bovine 58 kDa we have observed. These observations collectively support the idea that the complete VASAP-60 protein would migrate at 88 kDa, and that the 58-kDa and 50-kDa proteins could represent partial proteolytic fragments of the 88-kDa protein.
Comparison of VASAP-60 to the human 80K-H and murine ß-GII proteins reveals conservation of specific structural traits: 1) 17 cysteines clustered at the amino and carboxyl terminal ends, 2) two potential EF-hand calcium-binding domains, 3) a highly acidic domain flanked by proline-rich segments presenting putative Grb2-binding domain, 4) consensus sites for potential phosphorylation of serine and threonine residues, 5) a stretch of approximately 90 amino acids that is highly charged (116K to 211E), and 6) a potential ER targeting sequence. The high identity in amino acid composition, the similar isoelectric point (pI 4.2), and the conserved consensus protein motifs suggest that these proteins either represent members of the same protein family or are homologous proteins between species. The superfamily of EF-hand proteins are characterized by a common helix-loop-helix structure [37]. The EF-hand is known to coordinate calcium binding and is usually present in two to eight copies by which two domains composed of 12 amino acids each can interact [38]. Binding of calcium causes a conformational change that allows interaction with target proteins [39–41]. Since intracellular and intra-ER concentration of calcium is tightly controlled and plays important roles, it could be possible that VASAP-60 biological activity is controlled by levels of free calcium concentration. Goh et al. [9] and Kanai et al. [10] showed that 80K-H is phosphorylated in vivo on tyrosine residues. Searching for tyrosine residue phosphorylation consensus motifs using PROSITE [19] did not identify sites in VASAP-60. However, it is known that tyrosine residues flanked by negatively charged amino acids as observed in VASAP-60 (30, 54, 105, 268, 279, 364Y) may be candidate tyrosine kinase substrates [42]. The HDEL signal ER targeting sequence positioned at the carboxyl terminal end is well conserved between the VASAP-60, the human 80K-H, and the murine ß-GII. The HDEL sequence is similar to the KDEL sequence; both serve as protein retention signals in the ER and selectively retrieve protein from cis-Golgi back to the ER [40, 41, 43, 44]. These ER targeting sequences were shown to be functional in eukaryotes. Deletion or slight modification by mutagenesis of these signal sequences from ER resident proteins altered their subcellular localization into the secretory pathway and thereby resulted in their extracellular secretion. Conversely, appending the HDEL or KDEL signal at the carboxyl terminal end of a normally secreted protein caused the protein to be retained in the ER [40, 41, 45]. Since most known proteins bearing the KDEL or HDEL are soluble ER proteins, they must interact with a KDEL receptor, a seven transmembrane-spanning ER resident protein, to ensure their continuous retrieval from subsequent compartments (cis-Golgi) of the secretory pathway and their return to the ER [44, 46]. However, exceptions were found, as KDEL proteins were also observed in the secretory pathway, associated with the cytoplasmic membrane and secreted [47, 48].
Subcellular fractionation of proteins extracted from bovine luteal cells followed by Western blot analysis showed that VASAP-60 is associated with cellular membrane structures. Other groups have also reported the association of the human 80K-H with cellular membrane fractions [9, 10]. In an immunofluorescence study [8], the human 80K-H was associated with the surface cytoplasmic membrane of numerous cell types including mononuclear, endothelial, renal, and brain neuronal and glial cells interacting with AGE-specific receptors [8]. VASAP-60 was detected in a G/E fraction and in WGA purified proteins, indicating its association with intracellular membranes. While the relative importance of the endosomal and the Golgi apparatus was not studied in greater detail here, it was shown that VASAP-60 was present in internal membranes and associated at least in part with glycoproteins. It has recently been demonstrated that 80K-H interacts with the human fibroblast growth factor receptor III [10]. Thus our results on the subcellular localization of VASAP-60 with cellular membrane corroborate well with previous findings on the human 80K-H, and suggest that VASAP-60 may interact with other membrane-bound glycosylated proteins.
VASAP-60 presents an acidic domain composed of 21 aspartic and glutamic amino acids organized in tandem and flanked at its amino and carboxyl terminal ends by proline-rich stretches. This domain is located in the most hydrophilic region of VASAP-60 and may present potential protein-protein interacting binding sites. Acidic domains are known to mediate protein-protein interactions. Furthermore, the proline-rich stretches upstream (288YPPSPPAP) and downstream (301KEEQPPMP and 341KDAPPPAP) of the acidic domain present three minimal consensus motifs, PXXP, that are shared among all SH3 binding sites [48]. The SH3 binding sites can be divided in two classes according to the position of an arginine or lysine residue at the amino or carboxyl terminus, which determines the amino-to-carboxyl terminal ligand-binding orientation. The putative VASAP-60 SH3 binding sites would belong to class 1. However, the amino terminal arginine is one residue away from the optimal location found in most class 1 SH3 binding motifs (RXXPXXP). Meanwhile, flexibility in the distance between amino acids may exist in members of the class 1 ligands [49]. When the putative interacting protein/protein domain, SH3-acidic-SH3, is compared to the human 80K-H, murine ß-GII, or ß-GII-B1, only 63.9%, 52.3%, or 59.3% identity was found. Whether these structurally related proteins, bearing a divergent putative interacting protein/protein domain, represent members of the same protein family or orthologous proteins remains an open question. Comparison of the VASAP-60 gene structure with that of the human 80K-H [50], as well as the characterization of their respective interacting intracellular proteins, may help to answer this question. Collectively, the amino acid structure present in VASAP-60 supports the hypothesis that it would be involved in the formation of multimeric intracellular complexes.
Immunohistochemical staining of bovine tissues also revealed the presence of VASAP-60 in internal membrane, with a perinuclear and pericytoplasmic membrane staining associated with vacuoles/vesicles in specific cell types. VASAP-60 was localized in oocytes, granulosa and theca cells, luteal cells, and oviductal epithelial cells, while in the uterus it was associated with the glandular and epithelial endometrial cells. In the endometrial epithelial or glandular cells and in oviductal epithelial cells, VASAP-60 immunostaining was mainly located at their apical and basolateral sides, suggesting a polarity in its intracellular localization. In the kidney, VASAP-60 was associated with vacuoles or vesicles in the epithelial tubular cells. These cells are well known for their active uptake of proteins from the glomerular filtrate [51]. Thus, VASAP-60 is expressed at different levels in all the tissues we have analyzed so far, but its pattern of expression is related to specifically differentiated cells within a given tissue. Recently, it was postulated that the rat homologue protein could represent the noncatalytic ß subunit of GII [7]. GII is a glucose-trimming enzyme involved in the maturation of N-linked oligosaccharides that are added en block (dolichyl-PP-activated Glc-NAc2-Man9-Glc3) to specific asparagine residues in the nascent polypeptide. GII removed the two 1,3-linked glucose residues from the oligosaccharide residues, and it works in conjunction with other enzymes, namely glucosidase I, UDP Glc:glycoprotein glucosyltransferase, and calnexin [52], to induce the proper conformation of glycosylated proteins before they are released into the secretory pathway. The hypothetical role for the ß-GII was defined as that of an anchor for the catalytic subunit in the ER through the HDEL signal. The ß-GII was characterized during purification of CD45 interacting proteins [7]. The CD45 is a transmembrane tyrosine phosphatase associated with cytoplasmic membrane of leukocytes. In the process of GII purification, some groups reported the presence of an associated protein at 80 kDa or 58–60 kDa (cow [53], pig [34], rat [11, 54, 55]), whereas others did not [33, 35]. Furthermore, discrepancies were also found in the subcellular localization of GII. Electron microscopic studies in pig hepatocytes showed the presence of the enzyme in the ER [56], but the enzyme was also found associated with the cytoplasmic membrane and endosomes in pig kidney epithelial cells [57]. GII is believed to be a constitutive enzyme of all cells. If VASAP-60 or the human 80K-H is the postulated ß-GII [7], this does not explain why we have observed a differential cellular expression pattern for VASAP-60. Knowing that VASAP-60 has an ER retention signal, it is unclear why we detected its presence not only in association with the ER, as observed in the vicinity of the perinuclear region, but also with vacuoles close to the cytoplasmic membrane. The recent molecular characterization of pig GII [33] raises the question whether the 80 kDa is specifically related to GII. Clearly, further detailed studies are needed to clarify the role of VASAP-60 in the ER and/or other organelles of the vacuolar system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the "Fonds de la recherche scientifique médicale" (FRSM) of Belgium, the "Fonds pour la Formation de Chercheurs et l'Aide à la Recherche" (FCAR) of Quebec. F.R. was supported by the "Agence Francophone des Universités pour l'Enseignement Supérieur et la Recherche" (AUPELF-UREF). 
2 Correspondence: Jacques G. Lussier, Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, P.O. Box 5000, St-Hyacinthe, PQ, Canada J2S 7C6. FAX: 450 778 8103; lussij{at}medvet.umontreal.ca
Accepted: October 19, 1999.
Received: June 16, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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