Purification, cDNA Cloning, and Expression of a New Human Blood Plasma Glutamate Carboxypeptidase Homologous to N-Acetyl-aspartyl-alpha -glutamate Carboxypeptidase/Prostate-specific Membrane Antigen*

Richard GingrasDagger , Catherine RichardDagger , Mohamed El-Alfy§, Carlos R. Morales§, Michel PotierDagger , and Alexey V. PshezhetskyDagger

From the Dagger  Université de Montréal, Service de Génétique Médicale, Département de Pédiatrie, Hôpital Sainte-Justine, Montréal, Québec H3T 1C5, Canada and the § Department of Anatomy and Cell Biology, McGill University, Montréal, Québec H3A 2B2, Canada

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

We describe the identification, cDNA cloning, and biochemical characterization of a new human blood plasma glutamate carboxypeptidase (PGCP). PGCP was co-purified from human placenta with lysosomal carboxypeptidase, cathepsin A, lysosomal endopeptidase, cathepsin D, and a gamma -interferon-inducible protein, IP-30, using an affinity chromatography on a Phe-Leu-agarose column. A PGCP cDNA was obtained as an expressed sequence tag clone and completed at 5'-end by rapid amplification of cDNA ends polymerase chain reaction. The cDNA contained a 1623-base pair open reading frame predicting a 541-amino acid protein, with five putative Asn glycosylation sites and a 21-residue signal peptide. PGCP showed significant amino acid sequence homology to several cocatalytic metallopeptidases including a glutamate carboxypeptidase II also known as N-acetyl-aspartyl-alpha -glutamate carboxypeptidase or as prostate-specific membrane antigen and expressed glutamate carboxypeptidase activity. Expression of the PGCP cDNA in COS-1 cells, followed by Western blotting and metabolic labeling showed that PGCP is synthesized as a 62-kDa precursor, which is processed to a 56-kDa mature form containing two Asn-linked oligosaccharide chains. The mature form of PGCP was secreted into the culture medium, which is consistent with its intracellular localization in secretion granules. In humans, PGCP is found principally in blood plasma, suggesting a potential role in the metabolism of secreted peptides.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Cellular and secreted carboxypeptidases are important in the generation, processing, and inactivation of different vertebrate neuropeptides (for recent reviews, see Refs. 1-7). In particular, an important brain glutamate carboxypeptidase II (EC 3.4.17.21) also called N-acetyl-aspartyl-alpha -glutamate carboxypeptidase (NAALADase)1 (8, 9) hydrolyzes the neuropeptide, N-acetyl-L-aspartyl-L-glutamate, releasing glutamate, the dominant excitatory neurotransmitter/neuromodulator of the mammalian central nervous system (10, 11). NAALADase is also required for the intestinal uptake of folate and is responsible for the resistance of some tumors to methotrexate (12). Cloning of NAALADase (12-14) showed that it is identical to the so-called prostate-specific membrane antigen (PSMA), which is strongly expressed in prostate cancer and serves as a marker for detection of prostatic cancer metastasis (reviewed in Ref. 15). NAALADase is a 94-kDa transmembrane protein, homologous both to the transferrin receptor and the bacterial cocatalytic zinc metallopeptidases (16). Recent cloning of the human ileal membrane dipeptidyl peptidase, I100 (17), the catalytic domain of which is homologous to NAALADase/PSMA, revealed that in mammals, cocatalytic zinc metallopeptidases may be represented by multiple enzymes with different substrate specificity.

We describe a new human peptidase of the NAALADase/PSMA family, a 56-kDa blood plasma glycoprotein termed PGCP, which was affinity-co-purified with the lysosomal carboxypeptidase, cathepsin A (CathA; EC 3.4.16.1). CathA forms a high molecular weight complex with beta -galactosidase (EC 3.2.1.23), sialidase (neuraminidase; EC 3.2.1.18), and N-acetylgalactosamine-6-sulfate sulfatase (EC 3.1.6.4) that is essential for their function in the lysosome (18-20). About two-thirds of lysosomal CathA is not complexed with beta -galactosidase and sialidase (21) and can be excreted into plasma from platelets (22) and lymphoid cells (23). Our recent studies on CathA substrate specificity (24, 25) indicated that CathA is the major carboxypeptidase in human tissues able to cleave hydrophobic amino acid residues, including the amidated ones. In vitro, CathA can inactivate regulatory peptides like endothelin, substance P, bradykinin, and angiotensin I (22, 23, 26-29), suggesting that it can be involved in the regulation of central neural and cardiovascular functions. Our recent studies propose that CathA may be associated with the metabolism of peptides implicated in memory consolidation (30).

For purification of CathA, we designed an affinity column by coupling a specific substrate of CathA, Phe-Leu, to an epoxy-activated agarose matrix. Analysis of the protein fraction purified from human tissues using this column revealed a novel 56-kDa glycoprotein, PGCP. In this paper, we report cloning of PGCP cDNA; characterization of its primary structure, enzymatic activity, expression, and metabolism; and its immunolocalization in human tissues and cells, which altogether suggest that PGCP is a novel glutamate carboxypeptidase secreted into the blood plasma.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Purification of PGCP from Human Placenta-- A crude glycoprotein fraction was purified from 10 human placentas (~5 kg of wet tissue weight) using a concanavalin A-Sepharose affinity column (21). The preparation was passed through a p-aminophenyl-beta -D-thiogalactopyranoside-agarose affinity column to separate the beta -galactosidase-CathA-sialidase complex and then applied onto a 20-ml Phe-Leu-agarose affinity column, which specifically binds CathA and cathepsin D (CathD) (21). The column was washed with 20 volumes of 20 mM sodium acetate buffer, pH 4.75, containing 0.15 M NaCl, and eluted with 5 volumes of 0.1 M Tris-HCl buffer, pH 7.5, containing 5 mM benzyloxycarbonyl-Phe-Leu. The eluate was concentrated using an Amicone stirred cell with a PM-10 membrane, dialyzed against 10 mM Tris-HCl buffer, pH 7.5, centrifuged at 10,000 × g for 10 min, applied to an FPLC Mono Q column (Amersham Pharmacia Biotech) equilibrated with the same buffer, and eluted with a linear NaCl gradient from 0 to 0.4 M. The 0.075-0.1 M NaCl fractions, which contained cathepsin D and PGCP, were pooled, concentrated, applied to a FPLC Superose 12 gel filtration column (Amersham Pharmacia Biotech), and then eluted in 20 mM sodium acetate buffer, pH 5.2, containing 0.15 M NaCl and 0.02% (w/v) NaN3. Eluted fractions were analyzed by SDS-PAGE. Fractions corresponding to the first protein peak, containing only PGCP, were pooled, concentrated, and stored at -30 °C. The molecular masses of the eluted proteins were determined using the following molecular mass standards (Amersham Pharmacia Biotech): blue dextran (~2,000 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa). A similar procedure was used for the purification of PGCP from mouse liver.

N-terminal Amino Acid Sequencing of Proteins-- SDS-PAGE was performed as described by Laemmli (31) on an 11% acrylamide gel. Protein bands were stained with Coomassie Blue. For NH2-terminal sequencing, the proteins were electrotransferred to ImmobilonTM-P membrane (Millipore Corp.) in a Trans-Blot cell (Bio-Rad) for 2.5 h at 4 °C in 10 mM CAPS buffer, pH 11, 10% (v/v) methanol at a current of 480 mA. After staining with Coomassie Blue R-250, the bands were excised, and amino acid sequences were determined in an Applied Biosystems 470 A gas phase sequencer. Sequences were analyzed using the BLAST network service of the National Center for Biotechnology Information (Bethesda, MD).

EST Clone of PGCP-- The dbEST was screened with the NH2 terminal amino acid sequence of PGCP, DVAKAIINLAVYGKAQ(N)RSYERLALLVDTVG, using the NCBI pBLAST search engine. Clone 30142, which contained deduced amino acid sequence identical to the NH2-terminal sequence of PGCP (GenBankTM accession number R18560), was supplied by the IMAGE Consortium, Human Genome Center (Livermore, CA). The 1.62-kilobase pair PGCP cDNA insert was excised by HindIII/NotI digestion, subcloned into pBluescript (Stratagene), and sequenced using the PRISM Ready Reaction Dye Deoxy Terminator cycle sequencing kit on an Applied Biosystems 373A automated sequencer. Conditions for sequencing were 25 cycles at 96 °C for 30 s, 50 °C for 15 s, and 60 °C for 4 min. Products were purified by size exclusion chromatography on CENTRI SEP columns (Princeton Separations) and fractionated by electrophoresis on a 6% denaturing (8.3 M urea) polyacrylamide gel. Sequences were analyzed using the SeqED software.

cDNA Cloning of PGCP-- A 5'-PGCP cDNA subclone was obtained by 5'-rapid amplification of cDNA ends polymerase chain reaction from total human placenta RNA using a MarathonTM cDNA amplification kit (CLONTECH) and an oligonucleotide primer, 5'-CCAATCGCTCATAGGATCTGTTCTG-3', complementary to the PGCP cDNA sequence (nucleotides 178-202). The product was cloned into a pCRII vector (Invitrogen) and sequenced. Analysis of the amplified sequence and its comparison with sequence of EST clone 30142 and several other EST clones containing fragments of the PGCP cDNA showed that clone 30142 contained a complete cDNA of PGCP with the exception of the first nucleotide of the initiation ATG codon.

To complete a PGCP cDNA at 5', we amplified a 400-kilobase pair 5'-end fragment using a 5'-CGATAAGCTTGCGGCCGCACGAGGCATGAA-3' primer, which contains the PGCP initiation methionine codon (underlined) plus 25 nucleotides of upstream sequence with HindIII and NotI sites (boldface type), a 5'-ACCCAGGATGGCTATCTTAT-3' primer, and the pBluescript-PGCP vector as a template. The fragment was digested with HindIII and EcoRI and cloned into pBluescript-PGCP, from which the HindIII-EcoRI cassette had been excised, and the insert of the resulting construct (pBluescript-PGCPc) was completely sequenced on each strand as above.

RNA Isolation and Northern Blots-- Human skin fibroblasts from normal controls were cultured to confluency in Eagle's minimal essential medium (Mediatech, Washington, D. C.) supplemented with 10% (v/v) of fetal calf serum (MultiCell) and antibiotics. Total RNA was isolated from the cell pellet or from human placenta tissue by ultracentrifugation in a CsCl2 gradient as described by Maniatis et al. (32).

Purified RNA was analyzed by Northern blot as described (32). A HindIII/NotI PGCP cDNA fragment from clone 31259 was labeled with [32P]dCTP using the Oligolabeling Kit (Amersham Pharmacia Biotech) and used as a hybridization probe.

Antibodies-- Rabbit polyclonal antibodies against PGCP were prepared as follows. A 577-kilobase pair fragment of PGCP cDNA spanning codons 127-320 was obtained by EcoRI/BglII digestion. After blunting with Klenow fragment DNA polymerase, the fragment was inserted into the SmaI site of pGEX-2T vector (Amersham Pharmacia Biotech). The resulting plasmid was expressed in Escherichia coli 801-C to produce PGCP-glutathione S-transferase fusion protein. The fusion protein was purified from the bacteria homogenate by affinity chromatography on glutathione-Sepharose (Amersham Pharmacia Biotech) as described by the manufacturer. Purified fusion protein (1 mg), homogeneous by SDS-PAGE analysis, was used to immunize a rabbit. IgG fraction purified from the antiserum by ammonium sulfate fractionation was passed through a recombinant glutathione S-transferase-Sepharose column to absorb the anti-glutathione S-transferase-specific antibodies. The resulting antibody preparation was used in a dilution of 1:10,000 or 1:15,000 for Western blotting, 1:500 for immunoelectron, and 1:200 for immunofluorescent microscopy.

Western Blotting-- Cellular or tissue homogenates, concentrated cellular medium, blood serum, or purified preparation of PGCP, was subjected to SDS-PAGE and electrotransferred to a NITRO ME nitrocellulose membrane (Micron Separations Inc., Westboro, MA). The detection of protein bands cross-reacting with anti-PGCP antibodies was performed using the BM Chemiluminescence kit (Boehringer Mannheim) in accordance with the manufacturer's protocol.

The deglycosylation of proteins was performed by treatment of 20-µg samples with 2 units of endoglycosidase F (Sigma) in 20 mM sodium phosphate buffer, pH 7.0, containing 0.1% (w/v) SDS, 0.2% (w/v) dithiothreitol, and 0.5% (v/v) Triton X-100 for 17 h at 37 °C.

Expression of PGCP in COS-1 Cells-- The full-length PGCP cDNA was obtained by NotI restriction of pBluescript-PGCPc and cloned into the NotI site of pCMV expression vector (33), kindly provided by Dr. S. G. MacGregor and Dr. T. Caskey (Baylor College of Medicine, Houston).

COS-1 cells were transfected with pCMV-PGCP expression vector using Lipofectamine (Life Technologies Inc.) in accordance with the manufacturer's protocol. 24, 48, and 72 h after transfection, different peptidase activities and control beta -hexosaminidase activity were assayed in cell homogenates and medium.

Immunofluorescent Microscopy-- Cultured human skin fibroblasts of normal controls were fixed on the glass slides with acetone/methanol (4:1) at -20 °C, washed in phosphate-buffered saline, blocked for 1 h with 2% (w/v) bovine serum albumin in phosphate-buffered saline, incubated with anti-PGCP antibodies at a final dilution of 1:200 for 1 h at room temperature, washed with phosphate-buffered saline, and further incubated for 30 min with rhodamine-conjugated goat anti-rabbit IgG at a dilution of 1:100. Epifluorescent microscopy was performed using a Zeiss Axioskop microscope.

Immunoelectron Microscopy-- Cultured human skin fibroblasts were detached from the culture dishes with a rubber policeman, washed with Hanks' balanced salt solution, and fixed in 4% paraformaldehyde, 0.5% glutaraldehyde in 50 mM phosphate buffer, pH 7.5. The cell pellets were dehydrated in methanol and embedded in Lowicryl K4 M as described previously (34, 35).

Ultrathin Lowicryl sections were mounted on 300-mesh Formvar-coated nickel grids (Polysciences, Inc., Warrington, PA). Each section was incubated for 15 min in 20 mM Tris-HCl-buffered saline (TBS), containing 0.1% (v/v) Tween 20 as well as 15% (v/v) goat serum and for 30 min with anti-PGCP antibodies diluted 1:500 in TBS. The sections were then washed four times with TBS containing 0.05% (v/v) Tween 20, incubated for 15 min in TBS containing 15% (v/v) goat serum, and for 30 min with colloidal gold (10 nm)-conjugated goat anti-rabbit IgG (Zollinger Inc., Montréal, Québec, Canada). The sections were washed two times with TBS containing 0.05% (v/v) Tween 20 and two times with distilled water and counterstained with uranyl acetate followed by lead citrate (34, 35). Normal rabbit serum was used as a control. Electron micrographs were taken using a Philips 400 electron microscope (Philips Electronics, Toronto, Ontario, Canada).

Metabolic Labeling-- 48 h after transfection with pCMV-PGCP expression vector, COS-1 cells grown to confluency in 75-cm2 culture flasks (~106 cells) were washed twice with Hanks' balanced salt solution and then incubated for 2 h in methionine-free Dulbecco's modified Eagle's medium (Life Technologies) supplemented with L-glutamine and sodium pyruvate, and for 40 min in 5 ml of the same medium supplemented with [35S]methionine (DuPont), 0.1 mCi/ml. The radioactive medium was then removed, and the cells were washed twice with Hanks' balanced salt solution and chased at 37 °C in Eagle's minimal essential medium supplemented with 20% (v/v) fetal calf serum.

At the times indicated in the figures, the cells were placed on ice, washed twice with ice-cold phosphate-buffered saline, and then lysed for 30 min on ice in 1 ml of radioimmunoprecipitation assay buffer, containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 5 µg/ml leupeptin, and 0.1 mM alpha -toluenesulfonyl fluoride. The lysate was collected and centrifuged at 13,000 × g for 10 min to remove the cell debris.

Immunoprecipitation, Electrophoresis, and Detection of PGCP-- 1.0 ml of lysate was incubated for 4 h with preimmune serum at a final dilution of 1:20. Then the pellet obtained from 300 µl of Pansorbin cells (Calbiochem) was added, and the resulting suspension was incubated for 2 h at 4 °C, followed by centrifugation for 10 min at 13,000 × g. Supernatants were incubated overnight with the anti-PGCP antibodies in a 1:100 final dilution and then for 2 h at 4 °C with the pellet from 100 µl of Pansorbin cells and precipitated as above. The pellet was washed three times with 1 ml of radioimmunoprecipitation assay buffer. The antigens were eluted from the pellet by the addition of 100 µl of a buffer containing 0.1 M Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 0.2 M dithiothreitol, and 0.02% (w/v) bromphenol blue. The proteins were denatured by boiling for 5 min, and 50 µl of each sample were subjected to SDS-PAGE according to Laemmli (31). The molecular weights were determined with 14C-labeled protein markers (Amersham Pharmacia Biotech). The gels were fixed in acetic acid/isopropyl alcohol/water (10/50/40), soaked for 30 min in AmplifyTM solution (Amersham Pharmacia Biotech), vacuum-dried at 60 °C, and analyzed by autoradiography.

Immunoaffinity Column Purification of PGCP-- Immunoaffinity gel was prepared by coupling of 25 mg of anti-PGCP rabbit antibodies to 3 ml of BrCN-activated Sepharose 4B (Sigma) using the manufacturer's protocol. The PGCP-containing fraction of human plasma proteins obtained by ammonium sulfate precipitation (33-50% saturation) from 30 ml of serum was loaded on the immunoaffinity column. The column was washed by 10 ml of 50 mM sodium phosphate buffer, pH 7.5, containing 0.4 M NaCl, and eluted with 5 ml of 0.2 M glycine buffer, pH 2.3. The pH of collected 1-ml fractions was immediately adjusted to 7.5 by 1 M Tris-HCl buffer.

Enzyme Activity Assays-- CathA activity was measured as described by Pshezhetsky et al. (24) with benzyloxycarbonyl-Phe-Leu as a substrate; CathD activity was measured according to the method of Barret (36) with bovine hemoglobin (Sigma) as a substrate; and beta -hexosaminidase activity was measured using 4-methylumbelliferyl-glucosaminide (37). Aminopeptidase, dipeptidyl peptidase, endopeptidase, carboxypeptidase, and esterase activities were assayed fluorometrically or spectrophotometrically as described previously using the following substrates (all Bachem or Sigma): H-Ala-AMC, H-Arg-AMC, H-Glu-AMC, H-Leu-AMC, H-Lys-AMC, H-Phe-AMC, H-Gly-Phe-beta NA, Z-Arg-Arg-beta NA, benzoyl-DL-Arg-pNA, Z-Gly-Gly-Leu-pNA, Suc-Phe-Leu-Phe-pNA, Suc-Leu-Tyr-AMC, Z-Phe-pNA, FA-Ple-Ala, FA-Phe-Arg, FA-Phe-Glu, FA-Phe-Gly, FA-Phe-Phe, and FA-Glu-Tyr. Glutamate carboxypeptidase activity was measured with Ac-Asp-Glu (Sigma) substrate as follows. The 100 µl of incubation mixture contained 1 mM Ac-Asp-Glu and 1-5 µg of sample protein in 100 mM Tris-HCl buffer, pH 7.5, containing 1 mM ZnCl2. After 1-15 h of incubation at 37 °C, samples were incubated for 3 min at 100 °C and mixed with 0.9 ml of 100 mM Tris-HCl buffer, pH 8.0, containing 1 mM NAD+ (Sigma) and 0.25 units/ml of bovine glutamate dehydrogenase (Boehringer Mannheim). After a 1-h incubation at 37 °C, the concentration of glutamate was measured spectrophotometrically at 340 nm using the calibration curve obtained with 1-200 nmol of glutamate. One unit of enzyme activity corresponds to a conversion of 1 µmol of substrate/min. Proteins were assayed according to Bradford (38) with bovine serum albumin (Sigma) as a standard.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Purification and NH2-terminal Sequencing of PGCP-- The fraction of crude glycoproteins of human placenta retained by the Phe-Leu-agarose affinity column contained four major proteins. We separated these proteins by FPLC ion exchange chromatography using Mono Q column (Fig. 1) and identified three of them by NH2-terminal sequencing as cathepsin D (CathD) (peak I eluted at NaCl concentration between 0.057 and 0.1 M), CathA (peak II, 0.1-0.17 M NaCl), and a so-called IP-30 (39), a 30-kDa glycoprotein induced in hematopoietic cells in response to gamma -interferon, the biological function of which is unclear (peak III, 0.27-0.35 M NaCl). Peak I in addition to 31- and 15-kDa subunits of CathD also contained a 56-kDa glycoprotein, further termed PGCP. NH2-terminal amino acid sequence of PGCP, (G)DVAKAIINLAVYGKAQ(N)RSYERLALLVDTVG didnot match any of proteins in the Non-Redundant GenBankTM data base (all nonredundant GenBankTM cDNA sequence translations, plus all entries from the Protein Data Bak, Swiss-Prot, and PIR data bases).


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1.   FPLC anion-exchange chromatography on Mono Q column (a) and SDS-PAGE analysis (b) of the proteins purified from human placenta using concanavalin A-Sepharose and Phe-Leu-agarose affinity columns. Dashed line, absorbance at 280 nm; , CathA activity; open circle , CathD activity; dotted line, NaCl concentration. The indicated fractions were analyzed by SDS-PAGE, as described. The protein bands are identified on the right of the gel: 30- and 20-kDa polypeptide chains of CathA (CathA30 and CathA20); 31- and 14-kDa polypeptide chains of CathD (CathD31 and CathD14), gamma -interferon inducible protein, IP-30, and PGCP.

PGCP and CathD were resolved on a Superose 12 FPLC gel filtration column. Eluted fractions were analyzed for CathD activity and by SDS-PAGE (Fig. 2). Using a Superose 12 column, the molecular mass of PGCP (peak I at Fig. 2) was estimated as 120 kDa, suggesting that PGCP may form homodimers of 56-kDa subunits.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   FPLC gel filtration on Superose 12 HR column (a) and SDS-PAGE analysis (b) of the FPLC Mono Q chromatography fractions corresponding to peak I on Fig. 1a and containing PGCP and CathD. Dashed line, absorbance at 280 nm; open circle , CathD activity. The positions of the elution peaks of the molecular mass standards and void volume (V0) are shown by arrows. The indicated gel filtration fractions were analyzed by SDS-PAGE as described. The protein bands are identified on the right of the gel: 31-kDa polypeptide chain of CathD (CathD31) and PGCP.

cDNA and mRNA of PGCP and Its Chromosomal Localization-- A search in the dbEST data base showed that the NH2-terminal amino acid sequence of PGCP had a 100% identity with a deduced amino acid sequence of an EST clone 30142 (GenBankTM accession number R18560). From this cDNA fragment, which we extended by 5' rapid amplification of cDNA ends polymerase chain reaction, we derived a cDNA that we predict contains the entire PGCP coding sequence (see "Experimental Procedures"). This cDNA, confirmed by sequencing of several independent clones, contains a 1623-base pair open reading frame starting from a potential ATG initiation codon and 147 base pairs of 3'-untranslated region containing a polyadenylation site (Fig. 3). On Northern blots of mRNA from human placenta and fibroblasts, the PGCP cDNA hybridized with a single ~1.7-kilobase pair transcript, indicating that the acquired cDNA was near full-length (Fig. 4).


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 3.   Nucleotide sequence of PGCP cDNA and predicted amino acid sequence. The putative signal peptide is boxed, and the NH2-terminal amino acid sequence of the purified protein is underlined. Potential glycosylation sites are marked with black dots, and the terminal stop codon is marked with an asterisk.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Northern blot analysis of PGCP mRNA. 20 µg of total RNA from cultured human skin fibroblasts (lane 1) and placenta (lane 2) were used. The membrane was hybridized with 32P-labeled PGCP cDNA probe as described under "Experimental Procedures."

Analysis of the deduced 541-amino acid sequence of PGCP using a SignalP program (40) predicted that the first 24 amino acids may represent the signal peptide (boxed in Fig. 3), with a typical positively charged n-region (amino acids 1-3), a central h-region represented by a hydrophobic alpha -helix (amino acids 4-18), and a polar c-region with a predicted cleavage site between Cys24 and Lys25 (41). The NH2-terminal sequence of the purified protein (underlined in Fig. 3) starts at Asp45, suggesting that amino acids 25-45 may represent a propeptide not retained in mature PGCP. The sequence contains 5 potential N-glycosylation sites (indicated by black dots in Fig. 3), which is consistent with the binding of PGCP to concanavalin A-Sepharose.

Screening of the dbSTS library at GenBankTM with an accession number R18560 for the EST clone revealed the identity of PGCP with an unidentified STS transcript WI-6164 (similar to Cda01e07, or D8S1377E) encoded on human chromosome 8 between D8S257 and D8S270 markers (8q22.2 locus).

Expression and Metabolic Labeling of PGCP-- To study the synthesis and processing of PGCP, we cloned its full-length cDNA in the pCMV expression vector (33) and expressed it in COS-1 cells. 48 h after transfection, the total cell homogenate and concentrated cell medium were studied by Western blot using antibodies against a recombinant PGCP-glutathione S-transferase fusion protein. We detected a 56-kDa protein that cross-reacted with anti-PGCP antibodies in the homogenates of COS-1 cells transfected with pCMV-PGCP expression vector but not in untransfected control cells or cells transfected with pCMV-beta -galactosidase vector (Fig. 5a). The molecular size of the expressed protein was similar to that of PGCP purified from human placenta (Fig. 5a, lane 1), suggesting that in COS-1 cells PGCP is properly processed and glycosylated. Western blot analysis of the culture medium (Fig. 5b) showed that the majority of expressed PGCP is secreted. The treatment of the cells with 10 mM NH4Cl (19) did not increase the secretion of PGCP, suggesting that mannose 6-phosphate receptors are not involved in the trafficking of this protein.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Expression and biogenesis of PGCP in COS-1 cells. a, Western blot of homogenates of COS-1 cells harvested 48 h after transfection with pCMV-PGCP (lanes 2 and 3) or with pCMV-beta -galactosidase (lanes 4 and 5) or of homogenates of nontransfected cells (lanes 6 and 7). Lane 1 contained 0.5 µg of purified PGCP. Protein samples (20 µg for lanes 2, 4, and 6 and 10 µg for lanes 3, 5, and 7) were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and stained with rabbit anti-PGCP antibodies as described. b, Western blot of culture medium of COS-1 cells 48 h after transfection with pCMV-PGCP. Cells were cultured in the presence (lane 1) or in the absence (lane 2) of mannose 6-phosphate. 20 µg of total protein were applied on each lane. c, fluorographs of pulse labeling and chase of PGCP in COS-1 cells 48 h after transfection with pCMV-PGCP expression vector. Lane 1, pulse; lanes 2-5, chase for 1, 3, 6, and 22 h, respectively. Molecular mass values of PGCP precursor (62 kDa) and its mature form (56 kDa) are shown.

30-h pulse-chase experiments (Fig. 5c) demonstrated that PGCP is initially synthesized as a 62-kDa polypeptide, with a molecular weight consistent with that of the PGCP precursor. During a 6-h chase, the precursor is completely processed to a 56-kDa mature form similar to that detected by Western blotting in the cell lysates and in culture medium (Fig. 5, a and b).

Tissue and Cellular Distribution of PGCP-- Western blot analysis showed that the protein is unequally expressed in human tissues (Fig. 6a). PGCP was abundant in placenta and kidney; low in muscles, liver, and skin fibroblasts; and undetectable in brain or white blood cells. Since the highest level of PGCP was found in total blood, we performed a Ficoll fractionation of blood and found that the protein is localized exclusively in plasma (Fig. 6a).


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6.   Detection of PGCP in human tissues and blood fractions. a, Western blots of total homogenates of placenta (lane 1), total brain (lane 2), kidney (lane 3), muscles (lane 4), white blood cells (lane 5), fibroblasts (lane 6), liver (lane 7), total blood from fetal umbilical cord (lane 8), total venous blood (lane 9), blood plasma (lane 10), and red blood cells (lane 11). 20 µg of protein were applied on each lane. b, PGCP purified from human placenta or blood plasma before (-) or after (+) treatment with endoglycosidase F.

To characterize PGCP detected in blood plasma, we developed its rapid purification procedure using an immunoaffinity column. The fraction of blood serum proteins obtained by ammonium sulfate precipitation (33-50% saturation) was loaded on the immunoaffinity column containing anti-PGCP antibodies coupled to BrCN-activated Sepharose 4B. The preparation eluted from the column with 0.2 M glycine buffer, pH 2.3, contained only a 56-kDa protein that strongly cross-reacted with anti-PGCP antibody and had an affinity to the Phe-Leu-agarose column. Endoglycosidase F treatment of PGCP preparations purified from human placenta using a Phe-Leu-agarose column and from human blood using an immunoaffinity column equally reduced their molecular mass to 52 kDa (Fig. 6b), close to that predicted for the mature deglycosylated PGCP, suggesting that the processing and glycosylation of PGCP secreted into blood serum is similar to that of PGCP purified from placenta.

The intracellular distribution of PGCP was studied in skin cultured fibroblasts by both immunohistochemistry and immunoelectron microscopy. Fluorescent microscopy of fibroblasts revealed a peripheral punctate intracellular staining consistent with localization of PGCP in a visicular compartment (Fig. 7, upper panel). Indeed, immunoelectron microscopy of fibroblasts with anti-PGCP antibody (Fig. 7, lower panel), revealed heavily labeled electron lucent vesicles of approximately 150 nm in diameter that are located close to the plasma membrane. Taking into the account that PGCP is found in blood plasma and that most of PGCP from transfected COS-1 cells was secreted into the culture medium, the results of the immunofluorescent and immunoelectron microscopy can be interpreted as the localization of PGCP in secretory granules.


View larger version (66K):
[in this window]
[in a new window]
 
Fig. 7.   Localization of PGCP within cultured human skin fibroblasts by immunofluorescence (upper panel) and immunoelectron microscopy (lower panel). Upper panel, fibroblasts were fixed with cold acetone/methanol (4:1) and stained with anti-PGCP rabbit antibodies, followed by staining with rhodamine-conjugated goat anti-rabbit IgG antibodies. Magnification was × 400. Lower panel, a, electron luscent vesicles close to the plasma membrane are heavily labeled with 15-nm gold particles after incubation with anti-PGCP antibodies. b, the same vesicles are unreactive with normal rabbit serum (control). Magnification was × 24,700. Bar, 100 nm.

Amino Acid Sequence Homology of PGCP to Amino- and Carboxypeptidases-- The homology search in GenBankTM Non-Redundant data base and SwissProt data bases using the complete amino acid sequence of PGCP revealed significant homology with human, rat, and mouse NAALADase/PSMA as well as with bacterial zinc aminopeptidases from Aeromonas proteolytica (Vibrio proteolyticus, VIBR), Vibrio cholerae, and Streptomyces griseus (SGAP). Lower homology was found with X-Hex-dipeptidase (carnosinase) from Lactobacillus delbueckii subsp. lactis and a carboxypeptidase G2 (folate hydrolase G2) from Pseudomonas sp. (CPG2). Since the tertiary structures of VIBR and SGAP are resolved by x-ray crystallography (42-44) and the structure of human NAALADase/PSMA was recently modeled (16), we performed the alignment of the PGCP amino acid sequence with the sequences of these enzymes to study conservation of the residues that comprise the active site regions (Fig. 8). PGCP shows similarity to NAALADase/PSMA all through its sequence; the overall amino acid identity with human and rat PSMA is 27.1 and 25.0%, respectively (Table I). The lowest local identity score between PGCP and PSMA is observed for the sequence fragments suggested to be a NH2-terminal cytoplasmic "tail" (amino acids 1-18) and a transmembrane domain (amino acids 19-42) of PSMA protein. This is consistent with our finding that, unlike PSMA, PGCP is a soluble enzyme. The highest identity score was observed between the "catalytic domain" of PSMA (residues 279-587 in human and 276-589 in rat protein) and the aligning residues of PGCP (Table I and Fig. 8), suggesting that PGCP may have a similar catalytic domain spanning residues Gly218-Leu451. This fragment of PGCP also has the highest local identity with the VIBR and SGAP sequences (Table I).


View larger version (82K):
[in this window]
[in a new window]
 
Fig. 8.   Amino acid sequence alignment of PGCP with homologous peptidases: zinc aminopeptidases from A. proteolytica (V. proteolyticus, VIBR) and S. griseus (SGAP) or NAADAse/prostate-specific membrane antigen from humans (PSMAHUM) and rat (PSMARAT). The figure shows the central part of the deduced "catalytic domain" of PGCP (amino acids 255-428, ~30% of the total sequence) and aligning residues of PCMAHUM, PCMARAT, VIBR, and SGAP that demonstrated the highest identity score as is shown in Table I. Alignment was performed using the Lipman-Pearson algorithm and a BLOSUM62 matrix by ProteinManagerTM software package for sequence analysis (ACD Inc.). Identical amino acids are colored. Hyphens represent gaps introduced to optimize the alignment. Active site residues are indicated with the following symbols: black-diamond , nucleophyl; black-down-triangle , zinc-binding residues, ×, residues of the substrate-binding pocket. Numbers refer to the positions of amino acids.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Percentage of identical amino acids in sequences of PGCP and homologous cocatalytic metallopeptidases as aligned in Fig. 8
The values shown are percentages of identical amino acids in the whole sequences (upper right) or in the deduced "peptidase domains" (lower left) that included the following residues: Gly218-Leu451 of plasma glutamate carboxypeptidase (PGCP), Ala274-Leu587 of human and Ala276-Leu589 of rat NAADase/prostate-specific membrane antigen (PSMAHUM and PSMARAT, respectively), Ile110-Leu446 of zinc aminopeptidase from A. proteolytica (VIBR), and the whole sequence of zinc aminopeptidase from S. griseus (SGAP).

VIBR, SGAP, CPG2, and NAALADase/PSMA all belong to peptidase family M28, including amino- and carboxypeptidases, that are called cocatalytic because these peptidases contain two zinc atoms closely located in their catalytic site (16, 45). The first zinc atom is coordinated with Asp, Glu, and His, and the second is coordinated with His and two Asp residues. Since these residues are topologically conserved in all aligned proteins, we suggest that PGCP also contains two cocatalytic zinc atoms ligated with Asp302, Glu337, and His433 and with His290, Asp302, and Asn353, respectively (marked by triangles in Fig. 8). As in VIBR, SGAP, and NAALADase/PSMA, Asp302 can be a ligand for both zinc atoms. Catalytic site nucleophyl, Glu336, is also topologically conserved in PGCP and may form a cis-peptide bond with Glu337 as in the case of other members of the M28 family. In VIBR and SGAP aminopeptidases, the specificity pockets are formed by markedly hydrophobic residues (for example, Met280, Ile299, Cys329, Tyr331, Cys333, Met348, Phe350, Phe354, Tyr357, and Ile361 in VIBR). In contrast, only two of the corresponding positions are occupied by hydrophobic residues in PGCP (Ile254 and Leu269). Similarly to NAALADase, PGCP contains basic residues (Arg382, Lys428) in the substrate binding pocket that may contribute to the binding of substrates with negatively charged amino acids.

Carboxy- and Endopeptidase Activity of PGCP-- In order to verify if PGCP has a peptidase activity as predicted both by its amino acid homology with amino- and carboxypeptidases and its affinity to Phe-Leu-agarose, we tested the preparations of PGCP purified from human placenta, mouse liver, and blood plasma for the enzymatic activity against the number of substrates including those of aminopeptidase (H-Ala-AMC, H-Arg-AMC, H-Glu-AMC, H-Leu-AMC, H-Lys-AMC, and H-Phe-AMC), dipeptidyl peptidase (H-Gly-Phe-beta NA and Z-Arg-Arg-beta NA), endopeptidase and esterase (benzoyl-DL-Arg-pNA, Suc-Phe-Leu-Phe-pNA, Z-Gly-Gly-Leu-pNA, Suc-Leu-Tyr-AMC, and Z-Phe-pNA), and carboxypeptidase (FA-Phe-Ala, FA-Phe-Arg, FA-Phe-Glu, FA-Phe-Gly, FA-Phe-Phe, FA-Glu-Tyr, and Ac-Asp-Glu). The results of these experiments are shown in Fig. 9a). We observed a significant glutamate carboxypeptidase activity with Ac-Asp-Glu substrate (2.5-4.5 nmol/min/mg of protein) but also a low chymotrypsin-like activity with endopeptidase substrate, Suc-Leu-Tyr-AMC (0.04-0.07 nmol/min/mg of protein) and esterase substrate, Z-Phe-pNA (~0.02 nmol/min/mg of protein). We have chosen these three substrates to assay the activity in the homogenate and culture medium of COS-1 cells transfected with pCMV-PGCP expression vector. The results (Fig. 9b) showed a 6-8-fold increase of glutamate carboxypeptidase activity, assayed with Ac-Asp-Glu; a 1.5-2-fold increase of esterase activity, assayed with Z-Phe-pNA; and a 7-9-fold increase of the endopeptidase activity assayed with Suc-Leu-Tyr-AMC in cells transfected with pCMV-PGCP as compared with untransfected cells or with pCMV-beta -galactosidase-transfected cells.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 9.   Peptidase activities of purified preparations of PGCP and in transiently transfected COS-1 cells. a, PGCP purified from mouse liver, human placenta, or human blood plasma showed detectable endopeptidase activity toward Suc-Leu-Tyr-AMC (A, left axis), Suc-Phe-Leu-Phe-pNA (C, left axis), and Z-Gly-Gly-Leu-pNA (D, left axis); esterase activity toward Z-Phe-pNA (B, left axis); and glutamate carboxypeptidase activity toward Ac-Asp-Glu (E, right axis). Activity values measured with other substrates listed under "Experimental Procedures" were below the detection level (less then 1 pmol/min/mg). b, enzymatic activities toward Suc-Leu-Tyr-AMC (A, left axis), Z-Phe-pNA (B, left axis), and Ac-Asp-Glu (E, right axis) substrates were increased in the homogenates and in the culture medium of COS-1 cells 48 h after transfection with pCMV-PGCP expression vector (PGCP) as compared with those of control COS-1 cells (C) or cells 48 h after transfection with pCMV-beta -galactosidase (GAL). Cells were transfected, and peptidase activities were assayed as described under "Experimental Procedures." Values represent means ± S.D. of triplicate experiments.

In conclusion, we have cloned a new 56-kDa human blood plasma glycoprotein. Enzymatic analysis of the protein showed that it has a glutamate carboxypeptidase activity, consistent with its amino acid sequence homology with zinc cocatalytic metalloprotease, glutamate carboxypeptidase II (also known as N-acetyl-aspartyl-alpha -glutamate carboxypeptidase and PSMA). In contrast to glutamate carboxypeptidase II, PGCP also showed endopeptidase activity with substrates containing hydrophobic amino acid residues at the P1 and P1' positions similar to that of chymotrypsin or calpain. Although understanding the exact biological function of this new peptidase requires further characterization of its physiological substrates, the high level of PGCP observed in blood serum suggests that the protein may play an important role in the hydrolysis of circulating peptides.

    ACKNOWLEDGEMENTS

We are grateful to ACD Inc. (Toronto, Ontario, Canada) for providing the Protein ManagerTM software package for sequence analysis, Dr. R. Kenigsberg for the help in immunocytochemical studies, Dr. G. Mitchell and Dr. L. Ashmarina for helpful advice, and M. Patenaude for excellent secretarial assistance.

    FOOTNOTES

* This work was supported in part by grants from the Medical Research Council of Canada and from the Fonds de La Recherche en Santé du Québec (to A. V. P. and C. R. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF119386.

To whom correspondence should be addressed: Service de Génétique Médicale, Hôpital Sainte-Justine, 3175 Côte Sainte-Catherine, Montréal, Québec H3T 1C5, Canada. Tel.: 514-345-4931; Fax: 514-345-4801; E-mail: alex{at}justine.umontreal.ca.

    ABBREVIATIONS

The abbreviations used are: NAALADase, N-acetyl-aspartyl-alpha -glutamate carboxypeptidase or glutamate carboxypeptidase II; PSMA, prostate-specific membrane antigene; CathA, cathepsin A/protective protein; CathD, cathepsin D; FPLC, fast protein liquid chromatography; TBS, Tris-HCl-buffered saline; PAGE, polyacrylamide gel electrophoresis; AMC, 7-amido-4-methylcoumarin; FA, 3-(2-furyl)acryloyl; beta NA, beta -naphthylamide; pNA, p-nitroanilide; Suc, succinyl; Z, benzyloxycarbonyl; EST, expressed sequence tag; PGCP, plasma glutamate carboxypeptidase; CAPS, 3-(cyclohexylamino)propanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
  1. Ardaillou, R. (1997) Curr. Opin. Nephrol. Hypertens. 6, 28-34[Medline] [Order article via Infotrieve]
  2. Beinfeld, M. C. (1997) Life Sci. 61, 2359-2366[CrossRef][Medline] [Order article via Infotrieve]
  3. Bhoola, K. D., Figueroa, C. D., and Worthy, K. (1992) Pharmacol. Rev. 44, 1-80[Medline] [Order article via Infotrieve]
  4. Hook, V. Y. (1988) Cell Mol. Neurobiol. 8, 49-55[Medline] [Order article via Infotrieve]
  5. Costa, E., Mocchetti, I., Supattapone, S., and Snyder, S. H. (1987) FASEB J. 1, 16-21[Abstract/Free Full Text]
  6. Skidgel, R. A., and Erdös, E. G. (1998) Immunol. Rev. 161, 12-141
  7. Skidgel, R. A. (1992) J. Cardiovasc. Pharmacol. 20 Suppl. 9, S4-S9[Medline] [Order article via Infotrieve]
  8. Robinson, M. B., Blakely, R. D., Couto, R., and Coyle, J. T. (1987) J. Biol. Chem. 262, 14498-14506[Abstract/Free Full Text]
  9. Stauch-Slusher, B., Robinson, M. B., Tsai, G., Simmons, M. L., Richards, S. S., and Coyle, J. T. (1990) J. Biol. Chem. 265, 21297-21301[Abstract/Free Full Text]
  10. Blakely, R. D., and Coyle, J. T. (1988) Int. Rev. Neurobiol. 30, 39-100[Medline] [Order article via Infotrieve]
  11. Passani, L. A., Vonsattel, J. P., Carter, R. E., and Coyle, J. T. (1997) Mol. Chem. Neuropathol. 31, 97-118[Medline] [Order article via Infotrieve]
  12. Pinto, T., Sufoletto, B. P., Berzin, M., Quia, C. H., Lin, S., Tong, P., May, F., Mukherjee, and Heston, W. D. W. (1996) Clin. Cancer Res. 2, 1445-1451[Abstract]
  13. Carter, R. E., Feldman, A. R., and Coyle, J. T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 749-753[Abstract/Free Full Text]
  14. Bzdega, T., Turi, T., Wroblewdka, B., She, D., Chung, H. S., Kim, H., and Neale, J. H. (1997) J. Neurochem. 69, 2270-2277[Medline] [Order article via Infotrieve]
  15. Heston, W. D. W. (1997) Urology 49, 104-112[CrossRef][Medline] [Order article via Infotrieve]
  16. Rawlings, N. D., and Barrett, A. J. (1997) Biochim. Biophys. Acta 1339, 247-252[Medline] [Order article via Infotrieve]
  17. Shneider, B. L., Thevananther, S., Moyer, M. S., Walters, H. C., Rinaldo, P., Devarajan, P., Sun, A. Q., Dawson, P. A., and Ananthanarayanan, M. (1997) J. Biol. Chem. 272, 31006-31015[Abstract/Free Full Text]
  18. D'Azzo, A., Hoogeveen, A., Reuser, A. J., Robinson, D., and Galjaard, H. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4535-4539[Abstract]
  19. Hoogeveen, A. T., Verheijen, F. W., and Galjaard, H. (1983) J. Biol. Chem. 258, 12143-12146[Abstract/Free Full Text]
  20. Pshezhetsky, A. V., and Potier, M. (1996) J. Biol. Chem. 271, 28359-28365[Abstract/Free Full Text]
  21. Pshezhetsky, A. V., and Potier, M. (1994) Arch. Biochem. Biophys. 313, 64-70[CrossRef][Medline] [Order article via Infotrieve]
  22. Jackman, H. L., Tan, F. L., Tamei, H., Beurling-Harbury, C., Li, X. Y., Skidgel, R. A., and Erdös, E. G. (1990) J. Biol. Chem. 265, 11265-11272[Abstract/Free Full Text]
  23. Hanna, W. L., Turbov, J. M., Jackman, H. L., Tan, F., and Froelich, C. J. (1994) J. Immunol. 153, 4663-4672[Abstract/Free Full Text]
  24. Pshezhetsky, A. V., Vinogradova, M. V., Elsliger, M-A., El-Zein, F., Svedas, V. K., and Potier, M. (1995) Anal. Biochem. 230, 303-307[CrossRef][Medline] [Order article via Infotrieve]
  25. Elsliger, M-A., Pshezhetsky, A. V., Vinogradova, M. V., Svedas, V. K., and Potier, M. (1996) Biochemistry 35, 14899-14909[CrossRef][Medline] [Order article via Infotrieve]
  26. Itoh, K., Kase, R., Shimmoto, M., Satake, A., Sakuraba, H., and Suzuki, Y. (1995) J. Biol. Chem. 270, 515-518[Abstract/Free Full Text]
  27. Jackman, H. L., Morris, P. W., Deddish, P. A., Skidgel, R. A., and Erdös, E. G. (1992) J. Biol. Chem. 267, 2872-2875[Abstract/Free Full Text]
  28. Kawamura, Y., Matoba, T., Hata, T., and Doi, E. (1976) J. Biochem. (Tokyo) 81, 435-444
  29. Matsuda, K. (1976) J. Biochem. (Tokyo) 80, 659-669[Abstract]
  30. Ashmarin, I. P., Buzinova, E., Vinogradova, M., Potier, M., and Pshezhetsky, A. V. (1997) Neurosci. Res. Commun. 21, 153-162[CrossRef]
  31. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  32. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, pp. 7.6-7.10, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  33. MacGregor, G. R., and Caskey, C. T. (1989) Nucleic Acids Res. 17, 2365[Medline] [Order article via Infotrieve]
  34. Sylvester, S. R., Morales, C. R., Oko, R., and Griswold, M. D. (1989) Biol. Reprod. 41, 941-948[Abstract]
  35. Hermo, L., Wright, T., Oko, R., and Morales, C. R. (1991) Biol. Reprod. 44, 1113-1131[Abstract]
  36. Barret, A. J. (1970) Biochem. J. 117, 601-607[Medline] [Order article via Infotrieve]
  37. Rome, L. H., Garvin, A. J., Allietta, M. M., and Neufeld, E. F. (1979) Cell 17, 143-153[CrossRef][Medline] [Order article via Infotrieve]
  38. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
  39. Luster, A. D., Weinshank, R. L., Feinman, R., and Ravetch, J. V. (1988) J. Biol. Chem. 263, 12036-12043[Abstract/Free Full Text]
  40. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Protein Eng. 10, 1-6[Abstract]
  41. VonHeijene, G. (1986) Nucleic Acids Res. 14, 4683-4690[Abstract]
  42. Chevrier, B., Schalk, C., D'Orchymont, H., Rondeau, J-M., Moras, D., and Tarnus, C. (1994) Structure 2, 283-291[Medline] [Order article via Infotrieve]
  43. Chevrier, B., D'Orchymont, H., Schalk, C., Tarnus, C., and Moras, D. (1996) Eur. J. Biochem. 237, 393-398[Abstract]
  44. Greenblatt, H. M., Almog, O., Maras, B., Spungin-Bialik, A., Barra, D., Blumberg, S., and Shoman, G. (1997) J. Mol. Biol. 265, 620-636[CrossRef][Medline] [Order article via Infotrieve]
  45. Rowsell, S., Pauptit, R. A., Tucker, A. D., Melton, R. G., Blow, D. M., and Brick, P. (1997) Structure 5, 337-347[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.