Genomic organization, expression profile, and characterization of the new protein PRA1 domain family, member 2 (PRAF2)

Gene 371 (2006) 154 – 165 www.elsevier.com/locate/gene

Genomic organization, expression profile, and characterization of the new protein PRA1 domain family, member 2 (PRAF2) Crystal S. Fo 1 , Craig S. Coleman 1 , Christopher J. Wallick, Alex L. Vine 2 , André S. Bachmann ⁎ Cancer Research Center of Hawaii, University of Hawaii at Manoa, 1236 Lauhala Street, Honolulu, HI 96813, USA Received 14 September 2005; received in revised form 8 December 2005; accepted 8 December 2005 Available online 14 February 2006 Received by D.A. Tagle

Abstract PRA1 domain family, member 2 (PRAF2) is a new 19 kDa protein with four putative transmembrane (TM) domains. PRAF2 (formerly designated JM4) belongs to a new protein family, which plays a role in the regulation of intracellular protein transport. Recently, PRAF2 was found to interact with the chemokine receptor CCR5 [Schweneker, M., Bachmann, A.S., Moelling, K., 2005. JM4 is a four-transmembrane protein binding to the CCR5 receptor. FEBS Lett. 579, 1751–1758]. In order to further study the function and regulation of PRAF2, we determined its genomic structure and its protein expression pattern in normal and cancerous human tissues. PRAF2 encodes a 178-residue protein, whose sequence is related to PRAF1 (PRA1/prenylin) and PRAF3 (JWA/GTRAP3–18). The human PRAF2 gene contains three exons separated by two introns and is located on human chromosome Xp11.23. The recombinant PRAF2 protein was readily expressed in Schneider 2 (S2) insect cells, and the native protein was detected in human tissues with strong expression in the brain, small intestine, lung, spleen, and pancreas. The protein was undetectable in tissue of the testes. Strong PRAF2 protein expression was also found in human tumor tissues of the breast, colon, lung, and ovary, with a weaker staining pattern in normal tissues of the same patient. Our studies show for the first time that the CCR5-interacting PRAF2 protein is expressed in several human tissues with a possible function in ER/Golgi transport and vesicular traffic. © 2006 Elsevier B.V. All rights reserved. Keywords: Chemokine receptor CCR5; Comparative genomics; Immunohistochemistry; JM4; PRAF protein family; Tumor tissue

1. Introduction

Abbreviations: bp, base pairs; cDNA, DNA complementary to RNA; DAB, diaminobenzidine; EAAT3, excitatory amino acid transporter 3; ER, endoplasmic reticulum; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GTRAP3–18, glutamate transporter EAAC1 (EAAT3)-interacting protein 3–18; HRP, horseradish peroxidase; JM4, Jena-Muenchen 4; kDa, kilodalton; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PRA1, prenylated Rab acceptor 1; PRAF2, PRA1 domain family, member 2; siRNA, small interfering RNA; S2, Schneider 2; SDS, sodium dodecylsulfate; TM, transmembrane. ⁎ Corresponding author. Cancer Research Center of Hawaii, Natural Products and Cancer Biology Program, University of Hawaii at Manoa, 1236 Lauhala Street, Honolulu, HI 96813, USA. Tel.: +1 808 586 2962; fax: +1 808 586 2970. E-mail address: [email protected] (A.S. Bachmann). 1 Equal contributions. 2 Present address: Department of Biological Structure, School of Medicine, University of Washington, Seattle, WA 98195, USA. 0378-1119/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.12.009

PRA1 domain family, member 2 (PRAF2) is a new protein, which contains a large prenylated Rab acceptor 1 (PRA1) domain. The PRAF2-related proteins PRA1, JWA, GTRAP3– 18, and Arl6-IP5 also contain a PRA1 domain, and together, they form the new family of PRAF proteins. PRAF2 was previously designated Jena-Muenchen 4 (JM4) in the NCBI Protein Database (Accession No. NP_009144) and was shown to interact with the carboxy-terminus of chemokine receptor CCR5, a G protein-coupled receptor (GPCR), which serves as a co-receptor for the human immunodeficiency virus type 1 (HIV1) (Schweneker et al., 2005). Most recently, we found that PRAF2 interacts with GDE1/MIR16, a novel mammalian glycerophosphoinositol phosphodiesterase, using the yeast two-hybrid assay (Bachmann et al., 2006). In an unrelated study, the PRAF2 gene was identified by gene array as one of several hypoxia-regulated genes (Wykoff et al., 2004).

C.S. Fo et al. / Gene 371 (2006) 154–165

Some PRAF2-related proteins have been studied more extensively and functional data are available. For example, the glutamate transporter-associated protein 3–18 (GTRAP3– 18), which is the rat homologue of human JWA, was found to modulate the neuronal glutamate transporter EAAC1 by direct protein interaction (Lin et al., 2001a; Butchbach et al., 2002; Inoue et al., 2005). Another protein, which shares homology with PRAF2, is PRA1, the human homologue of the Yptinteracting yeast protein 3 (Yip3) (Sivars et al., 2003; Seabra and Wasmeier, 2004). PRA1 is associated with the Golgi membrane and interacts with Rab proteins, which belong to the Ras superfamily of low molecular weight GTP-binding proteins. These proteins are involved in intracellular trafficking (Martincic et al., 1997; Bucci et al., 1999; Liang and Li, 2000) and the interaction with PRA1 affects Rab membrane association and targeting (Lin et al., 2001b). PRA1 has also been shown to insert into the endoplasmic reticulum (ER) membrane, followed by vesicular transport along the exocytic pathway to the Golgi complex where it may regulate the functions of prenylated proteins (Liang et al., 2004). Arl6-IP5 is an interacting protein of the ADP-ribosylation like factor (Arl6), and also shares sequence homology with PRAF2. Like Rab proteins, the ADP-ribosylation factor (ARF) group of proteins belongs to the Ras superfamily (Ingley et al., 1999), and Arl-6 was recently identified as the gene underlying Bardet–Biedl syndrome type 3, a multisystemic disorder characterized by obesity, blindness, polydactyly, renal abnormalities, and cognitive impairment (Fan et al., 2004). PRAF2, PRA1, JWA, GTRAP3–18, and Arl6-IP5 are functionally and structurally related proteins, which localize to the ER, Golgi, and vesicular structures and form a new family of transport-associated protein, the PRAF family. To date, very little information is available on PRAF2. In the present study, we identified the gene structure and expression profile of PRAF2 in normal and cancerous human tissues and we characterized its topology as well as its relation to other PRAF family members. 2. Materials and methods 2.1. Reagents and antibodies Copper sulfate was purchased from Sigma (St. Louis, MO) and prepared as a 100 mM stock solution. A polyclonal peptide antibody against the carboxy-terminus of PRAF2 was raised in rabbits and the affinity-purified end product (600 μg/ml) used at 1 : 1000 dilution. As a negative control, the PRAF2 antibody was blocked/competed by pre-absorption with the carboxyterminal PRAF2 blocking peptide (same as used for rabbit immunization) using a five-fold excess (w : w) of blocking peptide in PBS (2 h at room temperature). Both the PRAF2 antibody and the blocking peptide are available from QED Bioscience (San Diego, CA). The peptide-blocked PRAF2 antibody (PRAF2-P) (12 μg/ml) was used at 1 : 20 dilution. The V5 epitope rabbit polyclonal antibody was from Chemicon (Temecula, CA) and used at 1 : 2000 to 1 : 5000 dilution. The βactin mouse monoclonal antibody was from Sigma (St. Louis,

155

MO) and used at 1 : 1000 to 1 : 2000 dilution, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (6C5) mouse monoclonal antibody was from Ambion (Austin, TX) and used at 1 : 10,000 dilution. The secondary anti-rabbit or antimouse antibodies conjugated to horseradish peroxidase (HRP) were from Amersham Biosciences (Piscatawa, NJ) and used at 1 : 5000 dilution. 2.2. PCR and plasmid construction The EST-DNA clone containing the PRAF2 gene in plasmid pOTB7 (ID 4297902) was purchased from Invitrogen (Carlsbad, CA) and used as template to PCR-amplify the cDNA of PRAF2. The primers PRAF2-fwd (5′-gacccagaattcatgtcggaggtgcggctgccaccg-3′) and PRAF2-rev (5′-cactcgctcgagggatccagcctcctgctcttgtcc-3′) were designed to include the restriction sites EcoRI and XhoI, respectively. In addition, a second reverse primer PRAF2-rev/stop (5′-cactcgctcgagctaggatccagcctcctgctcttgtcc-3′) was generated that contained a stop codon. The PCR reaction mixture contained 10 μl of 10× reaction buffer, 6 μl of MgCl2 (25 mM), 2 μl of dNTP mixture (10 mM), 2μl of each primer (10 pmol/μl), 10 μl of DNA template (10 ng/μl), and 0.5 μl of Taq DNA polymerase and was adjusted to a final volume of 100 μl with sterile water. The PCR was performed in a Mastercycler Gradient PCR machine (Eppendorf, Westbury, NY) according to the following schedule: pre-denature at 95 °C for 6 min, followed by 30 cycles at 95 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min. The two PCR products (one without and one with a stop codon at the 3′ end of PRAF2) were reamplified by PCR after they were recovered by agarose gel separation and gel extraction (Qiagen Inc., Valencia, CA) and individually cloned into the EcoRI and XhoI digested insect expression plasmid pMT/V5-His A (Invitrogen, Carlsbad, CA). This plasmid contains a metallothionein (MT) promoter for controlled induction of protein expression with copper sulfate and generates a V5 tag followed by a His6 tag at the carboxyterminus of the protein. DNA sequencing revealed that both PCR products were in-frame at their 3′ and 5′ ends, but were missing an internal fragment of the PRAF2 gene (162 and 99 bp, corresponding to amino acid residues 83–136 and 83–113 of PRAF2, respectively). Using two internal BamHI restriction sites of PRAF2, the deletion-containing fragments were excised and replaced with a BamHI-digested fragment of the original PRAF2 EST-DNA clone in pOTB7. After ligation, several clones were selected from each transformation, amplified, and analyzed by DNA sequencing to confirm the correct insertion and orientation of the internal fragments. In this way we generated two plasmids, pMT.PRAF2-V5/His and pMT.PRAF2 (including the stop codon), both of which contained the fulllength PRAF2 cDNA, one with and one without carboxyterminal tags. Both plasmids were used for further study in S2 insect cells. 2.3. Insect cell culture and stable transfection Schneider 2 (S2) insect cells (Drosophila melanogaster) were maintained in suspension at 28 °C in ambient air in

156

C.S. Fo et al. / Gene 371 (2006) 154–165

Schneider's medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS (Hyclone, Logan, UT) as previously described (Bachmann et al., 2004). To generate the stable cell lines S2-PRAF2-V5/His and S2-PRAF2, the plasmids pMT.PRAF2-V5/His and pMT.PRAF2 were individually co-transfected with the selection plasmid pCoBlast at a 20 : 1 (w : w) ratio into S2 cells using the calcium phosphate coprecipitation method according to the manufacturer's directions (Invitrogen, Carlsbad, CA). The transfected cells were selected by adding 25 μg/ml blasticidin (InvivoGen, San Diego, CA) to the medium. After 2 weeks of selection, the expression of recombinant PRAF2-V5/His and PRAF2 was induced by adding copper sulfate to the medium (0.5 mM final concentration), and cells from 60 mm culture dishes were harvested at different time points (days 1, 3, and 5) by centrifugation (1000 rpm for 3 min). PBS-washed cells were suspended in S2 lysis buffer and processed as explained in Section 2.5. 2.4. Mammalian cell culture and siRNA transfection The human embryonic kidney (HEK)-293 cells were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 medium (Biosource, Rockeville, MD) containing 10% heat-inactivated FBS (Invitrogen, Carlsbad, CA). Cells were seeded in 12-well plates in 1 ml of medium at 1 × 105 cells per well. Semiconfluent (∼50%) cells were transfected with the siGENOME™ SMARTpool® reagent (#M-019671-00) containing four siRNA molecules that target human PRAF2 (Dharmacon, Lafayette, CO) in the presence of siRNA SiLentFect transfection reagent (BioRad, Hercules, CA). Cells were harvested after 48 h in RIPA buffer as described in Section 2.5. Untreated cells and cells treated with transfection reagent alone served as controls. Duplicate wells were processed in two independent experiments. 2.5. Immunoblot analysis The HEK-293 cell lysates were prepared in RIPA buffer (20 mM Tris–HCl, pH 7.5, 0.1% sodium lauryl sulfate, 0.5% sodium deoxycholate, 135 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA). The insect cell lysates were prepared in S2 lysis buffer (50 mM Tris–HCl, pH 7.8, 150 mM NaCl, and 1% NP-40). Both buffers were supplemented with the Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). The total protein concentration of cleared cell lysates was determined using the protein assay dye reagent from BioRad and bovine serum albumin (BSA) as standard. Cell fractionations of S2 cells and HEK293 cells were performed with the Mem-PER® Eukaryotic Membrane Protein Extraction Kit (Pierce, Rockford, IL), which allows the enrichment of integral membrane proteins from cultured cells. The detergent-solubilized integral membrane proteins (hydrophobic fraction) and the cytosolic proteins (hydrophilic fraction) were separated through phase partitioning according to the manufacturer's directions. All protein samples were mixed with reducing loading buffer,

boiled, and equal amounts of total protein or equal volumes (for cell fractionation) analyzed by SDS–PAGE and Western blotting using the Mini-Protean III system (BioRad, Hercules, CA). The PVDF membranes were blocked in Blotto buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.1% (v / v) Triton X-100) containing 5% non-fat dry milk (Santa Cruz Biotechnology, Santa Cruz, CA), incubated with primary antibody at room temperature (1 h), washed multiple times in distilled water, and incubated with HRP-conjugated secondary antibody (1 h). A ready-to-use human tissues INSTA-Blot™ Immobilon membrane representing eleven different human tissues (∼10 μg total protein per lane) and was used according to the manufacturer's directions (EMD Biosciences, La Jolla, CA). All membranes were stained with the ECL or ECL Plus detection reagents (Amersham Biosciences, Piscatawa, NJ) and protein bands visualized on Kodak Bio Max XAR film. Membranes were stripped at 50 °C for 30 min with ECL stripping buffer (62.5 mM Tris–HCL, pH 6.7, 2% SDS, 100 mM 2-Mercaptoethanol) and sequentially probed. Quantification (siRNA experiments) was performed as described previously (Wallick et al., 2005) using a BioRad Fluor-S Multi Imager and Quantity One Quantitation Software, Version 4 (BioRad Laboratories, Hercules, CA). 2.6. Immunohistochemistry The human tissue array slides were deparaffinized in xylene, rehydrated with graded alcohols, and brought to distilled water. Slides were placed in a Tissue Tek II staining container filled with Target Retrieval solution (DakoCytomation, Carpinteria, CA) and placed in a microprocessor controlled bench top pressure chamber (Pascual) (DakoCytomation, Carpinteria, CA). After incubation at 125 °C for 5 min and depressurization for 20 min, the slides were allowed to cool undisturbed for 15 min. The slides were washed in distilled water, incubated in wash buffer for 5 min, quenched in hydrogen peroxide, and washed again twice in wash buffer before the primary antibody PRAF2 (1 : 1000) or peptideblocked antibody PFAF2-P (1 : 20) was applied for 30 min. The slides were then rinsed twice with wash buffer and incubated in Envision Plus Polymer (DakoCytomation, Carpinteria, CA). After repeated washes, slides were treated with chromagen (DAB Plus) (DakoCytomation, Carpinteria, CA) for 5 min, washed three times, and counter stained with hematoxylin (Gill No. 3) (Fisher Scientific, Pittsburgh, PA). Washed slides were blued in ammonia water and dehydrated in alcohol, cleared and mounted. The stained tissue slides were analyzed using a Zeiss Axioplan microscope (Carl Zeiss, Goettingen, Germany) equipped with a CCD camera (Photometrics, Tucson, AZ). 2.7. Computational analyses The PRAF2 gene structure information (exon–intron junctions) was obtained from the NCBI databank at http://www. ncbi.nlm.nih.gov (Entrez GeneID 11230). The primary amino acid sequence of PRAF2 (NCBI Protein Database Accession

C.S. Fo et al. / Gene 371 (2006) 154–165

No. NP_009144) was analyzed with several computer programs. The Dense Alignment Surface (DAS) transmembrane prediction server at http://www.sbc.su.se/∼miklos/DAS/ was used to generate a hydrophobicity profile indicating the putative transmembrane domains (Cserzo et al., 1997). The PSORT II server at http://psort.ims.u-tokyo.ac.jp/form2.html was used to confirm the hydrophobicity profile and to predict the subcellular localization (Horton and Nakai, 1997). The multiple sequence alignment and the phylogenetic tree were generated by sequence comparison of PRAF2-related proteins of different species using the Lasergene software and the MegAlign program (DNASTAR, Madison, WI). The ClustalW method was chosen for the alignment (Thompson et al., 1994). The motif search in PRAF2 was performed with the ScanSite server at http://scansite.mit.edu (Obenauer et al., 2003). Generic phosphorylation sites and kinase specific phosphorylation sites were predicted using the NetPhos 2.0 server (Blom et al., 1999) at http://www.cbs.dtu.dk/services/NetPhos/ and the NetPhosK 1.0 server (Blom et al., 2004) at http://www.cbs.dtu. dk/services/NetPhosK/, respectively. 3. Results and discussion 3.1. Genomic organization of human PRAF2 The PRAF2 gene product (formerly designated JM4) was identified as an interacting protein of chemokine receptor CCR5 (Schweneker et al., 2005) and was recently renamed PRA1 domain family, member 2 (PRAF2) by the HUGO Gene Nomenclature Committee (http://www.gene.ucl.ac.uk/ nomenclature/). In order to obtain more information about PRAF2, we analyzed its gene structure by computational analysis. We found that the PRAF2 gene contains three exons separated by two introns (Fig. 1), and this exon–intron organization is comparable to the PRAF2-related gene PRAF3 (GTRAP3–18) (Butchbach et al., 2002). However, while the sizes of the three human PRAF2 exons (195 bp for exon 1, 218 bp for exon 2, and 850 bp for exon 3) are nearly identical to those of PRAF3, the sizes of the two PRAF2 introns (1158 bp for intron 1 and 424 bp for intron 2) were significantly smaller than found for PRAF3 and comparable to those

157

reported for PRAF3 of the pufferfish F. rubripes (Butchbach et al., 2002). Our analysis further revealed that the PRAF2 gene maps to the human chromosome Xp11.23, a locus that carries several genes possibly implicated in cancer including SSX1–9 (synovial sarcoma, X breakpoint 1–9), HDAC6 (histone deacetylase), PAGE1/4 (P antigen family members 1 and 4; prostate associated), PIM-2 oncogene, TFE3 (transcritiopn factor binding to IGHM enhancer 3), GATA1 (globin transcription factor 1), and melanoma antigen family D (MAGED1). By comparison, the PRAF3 gene is located on human chromosome 3p14 and the short arm of chromosome 3 has been implicated in various adult cancers (Kok et al., 1997). The PRAF2 gene produces a 178-residue protein with a predicted molecular mass of 19.3 kDa, an isoelectric point (pI) of 9.21, and a net charge of + 7.4 at pH 7.0. PRAF2 contains a large PRA1 domain (Fig. 1), which is found in several PRAF2-related proteins and derives its name from the protein prenylated Rab acceptor 1 (PRA1) (prenylin) (Martincic et al., 1997; Bucci et al., 1999; Liang and Li, 2000; Lin et al., 2001b). PRA1 is also referred to as PRAF1, and accordingly, the PRA1 domain-containing protein JWA/ GTRAP3–18 was renamed PRAF3, together forming the new family of PRAF proteins as listed in Table 1. 3.2. Evolutionary relationship of PRAF protein family members To gain more insights into the evolutionary relationship of this new PRAF protein family, we performed a multiple sequence alignment between PRAF1, PRAF2, and PRAF3 protein sequences of different species (Fig. 2A). The alignment revealed the two regions “NLLYYQTNY” and “HASLRLR”, both of which are highly conserved. These regions were previously identified by analyzing PRAF3 (GTRAP3–18) sequences from different species (Butchbach et al., 2002). Our alignment also revealed a total of eight residues, which are 100% identical from yeast (Saccharomyces cerevisiae) to man (Homo sapiens) (black boxes). To further study the interrelationship between PRAF proteins, the sequence alignment data were used to create a phylogenetic tree (Fig. 2B). The branches clearly show the formation of three groups

Fig. 1. Genomic structure analysis of human PRAF2. Schematic diagram of the exon–intron organization of the PRAF2 gene in the human genome and PRAF2 protein sequence. The genomic sequence information (exon–intron junctions) of the PRAF2 gene was obtained from the NCBI database at http://www.ncbi.nlm.nih.gov (Entrez GeneID 11230). The PRAF2 gene is located on chromosome Xp11.23. Exons and introns are indicated as white boxes and thick black lines, respectively. The nucleotides at each exon–intron junction are depicted in upper and lower case, respectively. The positions of the translation start codon (ATG) within exon 1 and the translation stop codon (TAG) within exon 3 are indicated with black arrows. The amino acid residues at the exon–intron junctions are indicated. The encoded PRAF2 protein is 178 amino acids (aa) in length and contains a PRA1 domain.

158

C.S. Fo et al. / Gene 371 (2006) 154–165

Table 1 The new PRAF protein family New symbol

Alternative symbols

NCBI protein database a

References b

PRAF1

PRA1

Q9UI14

PRAF2

Prenylin Yip3 JM4

Q9Z0S9 NP_014354 NP_009144

DXImx39e Sfc20 JWA GTRAP3–18 Arl6-IP5 Addicsin D2096.2 CG10373-PA DERP11 HSPC127

AAF66950 AAH64757 O75915 Q9ES40 AAM28950 BAD83603 T15897 AAF53696 BAC66462 AAF29091

Liang and Li, 2000 Liang et al., 2004 Sivars et al., 2003 Schweneker et al., 2005 N/A N/A Lin et al., 2001b Lin et al., 2001b Ingley et al., 1999 Inoue et al., 2005 N/A N/A N/A N/A

PRAF3

a Other accession numbers for the listed alternative symbol may be retrieved at the NCBI Protein Database (http://www.ncbi.nlm.nih.gov/). b Selected references, which refer to alternative symbols. N/A, no reference available.

representing PRAF1, PRAF2, and PRAF3, and each group further divides the protein subgroups corresponding to the taxonomic classification of the analyzed species. The sequence alignment and the phylogenetic tree support the classification of the new PRAF protein family and imply that these proteins are evolutionary conserved.

substrate, and regulated by phosphorylation. To search for specific kinase candidates, we used NetPhosK 1.0 and found that PRAF2 may be phosphorylated at S97 by protein kinase A (PKA) (score 0.68) and/or protein kinase C (PKC) (score 0.59). Additional predicted phosphorylation sites were: S139 for PKA (score 0.58), T161 for p38MAPK (score 0.54), and S70 for cdc2 (score 0.5). Phosphorylation sites and kinases, which were identified at medium or low stringency conditions (scores b 0.5), were excluded. We next used the DAS transmembrane prediction program to generate a hydrophobicity profile of PRAF2 (Fig. 3B). The profile revealed the presence of four hydrophobic segments (peaks), suggesting that PRAF2 contains four putative transmembrane (TM) domains (TM1–TM4). This result was confirmed with a second sequence analysis program (PSORT II) and proposes that PRAF2 is either an integral membrane protein or a membrane-associated protein. The PSORT II analysis further suggested that the membrane topology of PRAF2 is of type 3b and that the protein is predominantly (55.6%) associated with the ER. The collected computational data were then combined and converted to a topography model as illustrated in Fig. 3C. This model predicts four transmembrane domains with both amino- and carboxy-termini directed towards the cytoplasm. Also depicted is the location of the two conserved regions “NLLYYQTNY” and “HASLRLR” (gray circles, corresponding with gray boxes in Fig. 3A). The position of the two putative phosphorylation sites Y43 and S97 are highlighted (black circles) and are located at the amino-terminus and the intracellular loop of PRAF2, respectively.

3.3. Sequence analysis and topology of human PRAF2 3.4. Recombinant expression of PRAF2 in S2 insect cells We next performed a more detailed analysis of the PRAF2 gene and we found an ATG start codon (+ 17) and a TAG stop codon (+ 551) followed by the 3′ UTR, which contains one polyadenylation signal (AATAAA) and three ATTT domains (Fig. 3A). Similar sequence elements were identified in the PRAF3 gene (Butchbach et al., 2002). The ATTT domains were suggested to be important in the destabilization of mRNA (Shaw and Kamen, 1986; Butchbach et al., 2002). Computational analysis of the PRAF2 protein revealed with high probability an amphiphysin Src homology 3 group (SH3) domain using the ScanSite program at high stringency settings (Fig. 3A; boxed in white with solid lines). Amphipysins are brain-enriched proteins implicated in clathrin-mediated endocytosis that interact with the GTP-binding protein dynamin through their SH3 domains (Wigge et al., 1997; Owen et al., 1998; Evergren et al., 2004). Since dynamin is involved in the internalization of GPCRs by forming a complex with β-arrestin and Src, it is possible that PRAF2 serves as an adaptor protein between CCR5 (and possibly other chemokine receptors), dynamin, and β-arrestin. Using NetPhos 2.0 at high stringency settings, we identified two putative phosphorylation sites at tyrosine 43 (Y43) and serine 97 (S97) (Fig. 3A; circled and boldface). Of note, Y43 is one of the eight highly conserved residues depicted in Fig. 2A. These findings suggest that PRAF2 is a possible kinase

To examine the PRAF2 protein in vivo, we amplified the human PRAF2 cDNA by PCR and cloned the full-length gene into an insect cell expression plasmid. S2 insect cells can be cultured in suspension, stably transfected with DNA plasmids, and recombinant protein expression can be induced by the addition of copper sulfate to the culture medium. Using this S2 cell system, we generated two stable cell lines, S2-PRAF2-V5/His and S2-PRAF2, and induced the expression of recombinant PRAF2-V5/His (fulllength PRAF2, fused to a V5 tag and a His6 tag at its carboxy-terminus) or PRAF2 (full-length PRAF2 without carboxy-terminal tags) for 1, 3, and 5 days. As shown in Fig. 4A, PRAF2-V5/His and PRAF2 were not detected in S2 cells in the absence of copper sulfate, but were strongly expressed as early as 1 day after induction with copper sulfate. As expected, the V5 epitope-directed antibody only recognized PRAF2-V5/His (∼21 kDa, including tags), but not PRAF2 (~19 kDa, without tags) (Fig. 4A; upper panel). In contrast, the PRAF2-recognizing rabbit polyclonal antibody, which is directed against the carboxy-terminal end of PRAF2, detected PRAF2, but not PRAF2-V5/His (Fig. 4A; middle panel), thus suggesting that the V5/His tags block the PRAF2 antibody interaction with the carboxy-terminus of PRAF2. As a control, we treated the

C.S. Fo et al. / Gene 371 (2006) 154–165

159

Fig. 2. Evolutionary relationship between members of the PRAF protein family. (A) Multiple sequence alignment (ClustalW) was performed with amino acid sequences of PRAF proteins from different eukaryotic species. Two conserved regions are boxed. Residues, which are identical in all listed species, are blocked in black. The sequences were retrieved from the NCBI Protein Database. (B) The rooted phylogenetic tree shows the evolutionary relationships predicted from the multiple sequence alignment. The length of each pair of branches represents the distance between sequence pairs. The scale indicates the number of “Nucleotide Substitutions”.

PRAF2 antibody with blocking peptide (P) and reprobed the membrane with neutralized (inactive) PRAF2 antibody (PRAF2-P). The peptide-blocked PRAF2 antibody did not recognize PRAF2 (Fig. 4A; lower panel), confirming that the

generated antibody specifically detects PRAF2. Weak multimer formation of both PRAF2-V5/His and PRAF2 was found even under reducing conditions, a phenomenon that was previously observed (Lin et al., 2001a; Butchbach et al., 2002;

160

C.S. Fo et al. / Gene 371 (2006) 154–165

C.S. Fo et al. / Gene 371 (2006) 154–165

161

Fig. 4. Expression of recombinant PRAF2 proteins in Schneider 2 (S2) insect cells. (A) The PRAF2 gene (in the absence and presence of a stop codon) was cloned into the insect expression vector pMT/V5-His A, followed by cell transfection as described in Section 2.3. Two stable S2 cell lines (S2-PRAF2-V5/His and S2-PRAF2) were either left untreated (“−”) for 5 days or induced (“+”) with 0.5 mM copper sulfate for 1, 3, and 5 days. Cell lysates (10 μg total protein per lane) were separated by 15% SDS–PAGE and probed for PRAF2-V5/His and PRAF2 by Western blotting using the V5 epitope-directed antibody (upper panel), the PRAF2-recognizing rabbit polyclonal antibody (middle panel), the peptide-blocked PRAF2 antibody (PRAF2-P) (lower panel) or the β-actin recognizing antibody (bottom panel). B) S2PRAF2-V5/His and S2-PRAF2 cells were induced with 0.5 mM copper sulfate for 3 days and the cell lysates fractionated by phase partitioning. Cells in the absence of copper sulfate served as controls. The hydrophilic protein fraction (H) and the solubilized membrane protein fraction (M), derived from 5 × 106 cells, were volumeadjusted (to 300 μl each) and the relative amount of PRAF2-V5/His and PRAF2 in each fraction compared based on equal volumes. Equal fraction samples (5 μl per lane) were resolved by 15% SDS–PAGE and electroblotted membranes probed as described in (A).

Schweneker et al., 2005). Whether these high molecular PRAF2 complexes are of physiological relevance is currently not clear. To study the potential association of recombinant PRAF2 with cellular membranes, we separated cytosolic proteins (hydrophilic fraction) and integral membrane proteins (hydrophobic fraction) of S2-PRAF2-V5/His cells and S2-PRAF2 cells 3 days after induction and analyzed equal volumes of each fraction separately for the presence of PRAF2-V5/His and

PRAF2 by Western blot (Fig. 4B). As a control, non-induced cells were processed under identical conditions. PRAF2-His/V5 as well as PRAF2 were predominantly present in the membrane fraction (M) of induced cells but were also detected in the hydrophilic fraction (H). Identical to our observation shown in Fig. 4A, the V5 epitope-directed antibody recognized PRAF2V5/His, but not PRAF2 that lacks the carboxy-terminal tag (Fig. 4B; upper panel) while the PRAF2-recognizing antibody detected PRAF2, but not PRAF2-V5/His (Fig. 4B; middle

Fig. 3. Human PRAF2 cDNA sequence. (A) Complete nucleotide sequence of human PRAF2 (NCBI, GenBank Accession No. NM_007213). The deduced protein sequence is shown below. The predicted PRAF2 protein is 178 residues in length with a molecular size of ∼19 kDa. Two highly conserved domains are boxed in gray with solid lines. A predicted amphiphysin Src homology 3 group (Amphi-SH3) domain is boxed in white with solid lines. Two putative phosphorylation sites (Y43 and S97) are circled and boldface. Four putative transmembrane (TM) domains are underlined. The start codon (ATG) and the stop codon (TAG) are boxed. The ATTT regions of the 3' UTR are boxed with dashed lines. The polyadenylation signal is boxed with dashed lines and boldface. (B) Hydrophobicity profile prediction of PRAF2. The dotted line and the solid line indicate the cut-off at 1.7 (low stringency) and 2.0 (high stringency), respectively. The Dense Alignment Surface (DAS) program predicts four putative TM domains. (C) Topography model based on the computational predictions in (A) and (B). The four putative TM domains are connected through an intracellular loop. Amino- and carboxy-termini are directed towards the cytoplasm. The conserved domains and putative phosphorylation sites are indicated with gray and black circles, respectively.

162

C.S. Fo et al. / Gene 371 (2006) 154–165

panel). Incubation of the membrane with peptide-blocked PRAF2 antibody prevented the detection of PRAF2 (Fig. 4B; lower panel). As expected, cytoplasmic actin was only present in the hydrophilic fraction and was not detected in the membrane fraction (Fig. 4B; bottom panel). These results suggest that recombinant PRAF2-V5/His and PRAF2 are bound to cellular membranes of S2 cells, but are also available in soluble form in the cytoplasm. It is possible that PRAF2 assumes two structural conformations, one in which its transmembrane domains are exposed allowing its integration with membranes, and one in which the transmembrane domains are buried/camouflaged resulting in a more soluble, hydrophilic protein. Such conformational changes may be the result of posttranslational modifications as well as direct physical associations with interacting proteins and other cofactors. 3.5. Expression and siRNA downregulation of endogenous PRAF2 in mammalian cells To this date, the PRAF2 protein has only been analyzed under overexpressed cell culture conditions (Schweneker et al., 2005). Although these studies provide strong evidence for the existence of PRAF2, they do not provide full proof that mammalian cells produce native PRAF2 protein at endogenous levels. To answer this question, we used our new PRAF2 antibody, which successfully recognized recombinant PRAF2 in S2 insect cells (Fig. 4). We found that human embryonic kidney 293 (HEK-293) cells express endogenous PRAF2 (Fig. 5A; upper panel). To confirm that the identified band at ∼19 kDa is representative of PRAF2 we included two controls. First, we treated HEK-293 cells with PRAF2 siRNA for 48 h at three different concentrations (30–90 nM), which resulted in a dose-dependent downregulation of endogenous PRAF2 (Fig. 5A; upper panel). The quantification of these results revealed

that PRAF2 expression was reduced by 60% (n = 4) using siRNA at 90 nM. Then, the same membrane was reprobed with peptide-blocked PRAF2-P. Under these conditions PRAF2 was not detected (Fig. 5A; middle panel). The membrane was then probed for β-actin to confirm equal loading of proteins (Fig. 5A; lower panel). These results confirm that the size of native PRAF2 protein is ∼19 kDa as predicted, and they show for the first time that the protein is expressed in mammalian cells at physiological levels. To further examine the endogenous localization of PRAF2, we separated cytosolic proteins and integral membrane proteins of HEK-293 cells as described for S2 cells (Fig. 4B). Our data revealed that the majority of endogenous PRAF2 is located in the membrane fraction (M) (Fig. 5B; upper panel). The peptideblocked PRAF2 antibody (PRAF2-P) did not recognize the 19 kDa band confirming that the detected protein is PRAF2. As expected, cytoplasmic β-actin was present in the hydrophilic fraction, with minimal presence in the membrane fraction, the latter of which is likely due to a small cross-contamination of the cytosolic proteins into the membrane fraction. This result suggests that endogenous PRAF2 at physiological levels is mainly associated with cellular membrane structures, such as cell surface membranes and/or intracellular membranes, for example, of the ER, Golgi apparatus, and the nucleus. 3.6. Expression of PRAF2 in human tissue We next examined the distribution of native PRAF2 protein in a variety of human tissues by immunoblot analysis using the PRAF2 antibody (Fig. 6). PRAF2 was expressed in most tissues with a strong presence in the brain, small intestine, lung, spleen, and pancreas. The protein was not detected in tissue of the testes. In some instances, the formation of dimers was observed, however, the size varied

Fig. 5. Detection and siRNA downregulation of endogenous PRAF2 in mammalian cells. (A) Human embryonic kidney 293 (HEK-293) cells were treated with transfection reagent and PRAF2 siRNA (30–90 nm) for 48 h. Untreated cells and cells treated with transfection reagent alone served as controls. Equal amounts of total protein (10 μg per lane) were separated by 10% SDS–PAGE, electroblotted, and the same membrane sequentially probed with PRAF2 antibody (upper panel), peptideblocked PRAF2 antibody (PRAF2-P) (middle panel), and β-actin antibody (lower panel). Duplicate samples were processed in two independent experiments (n = 4). (B) The hydrophilic protein fraction (H) and the solubilized membrane protein fraction (M), derived from 5 × 106 HEK-293 cells, were volume-adjusted (to 300 μl each) and the relative amount of endogenous PRAF2 in each fraction compared based on equal volumes. Equal fraction samples (5 μl per lane) were resolved by 15% SDS–PAGE and electroblotted membranes probed as described in (A). The data in (B) are representative of four independent experiments with nearly identical results.

C.S. Fo et al. / Gene 371 (2006) 154–165

163

between individual tissues. This suggests that factors other than dimerization are responsible for the protein size, such as posttranslational modifications or the expression of PRAF2 isoforms. The membrane was also probed with peptideblocked PRAF2 antibody (PRAF2-P) and the result confirmed that the observed bands in Fig. 6 are specific and represent native PRAF2 (not shown). As expected, different tissues expressed various levels of β-actin and GAPDH, despite equal loading of the tissue lysates (10 μg total protein per lane). For example, it is known that β-actin is not expressed in adult cardiac and skeletal muscles. This result shows for the first time that native PRAF2 is expressed in various human tissues. 3.7. Expression of PRAF2 in human cancer tissue

Fig. 6. Native PRAF2 protein expression profile in various human tissues. A ready-to-use human tissue INSTA-Blot™ membrane (10 μg total protein per lane) was incubated with PRAF2 antibody and developed using the ECL detection method. PRAF2 monomer (∼19 kDa) was expressed in nearly all tissues except for the testes. Possible dimer formation and/or posttranslational modifications were observed in several tissues. Incubation of the same membrane with the peptide-blocked PRAF2 antibody (PRAF2-P) prevented binding to PRAF2, indicating the detected bands are specific and represent endogenous PRAF2 protein (not shown). As expected, β-actin and GAPDH were expressed in most human tissues, but at different concentrations.

Finally, the expression of PRAF2 protein was determined in human cancer tissues and compared to corresponding normal tissues. The tissue samples were obtained from four low-density tissue microarrays and analyzed by immunohistochemistry. Slides of four different tissue arrays were used and contained breast, colon, lung or ovarian tissue. Each tissue array contained spots derived from cancerous tissue, immediate adjacent tissue, and normal tissue (16 patients and 48 tissue spots per slide). The definition of a normal tissue is tissue that is procured at least 20 cm from the location of the tumor. All arrays contained cancerous and normal tissues derived from the same cancer patient except for the ovarian tissue array in which cancer

Fig. 7. Elevated expression of PRAF2 in human tumor tissues. Representative tissue samples are shown from patients with breast cancer (A), colon cancer (B), lung cancer (C), and ovarian cancer (D). Tissue immediately adjacent to the tumor and normal tissue of the same patient was included for comparison (A–C). In ovarian tumor tissue, stage I/II and stage IV tissue samples and normal tissue samples are not from the same patient (D). Tissue samples are from four tissue microarrays (Invitrogen, Carlsbad, CA). Individual slides (16 patients and 48 tissue samples per slide) of each array were stained with PRAF2 antibody or peptide-blocked PRAF2 antibody (PRAF2-P). Representative images from each slide are shown. Brown coloration indicates the expression of PRAF2. Nuclei appear blue in color.

164

C.S. Fo et al. / Gene 371 (2006) 154–165

tissues at different stages were compared with normal tissue from different patients. As shown in Fig. 7, PRAF2 was strongly expressed in tumor tissues of the breast, colon, lung, and ovary as well as tissues immediately adjacent to the corresponding tumor tissue. Expression of PRAF2 in corresponding normal tissues was significantly weaker, suggesting that PRAF2 is overexpressed in the examined cancerous tumors. Also of note was the observation that PRAF2 in tumor- and tumor-adjacent tissues was mainly localized in the cytoplasm, while in normal tissues, the protein appeared associated with cell nuclei, suggesting that PRAF2 is translocated in hyperproliferative tissues. Tissue samples treated with peptide-blocked PRAF2 antibody (PRAF2P) did not show brown staining patterns, thus confirming that the observed immunoreactivity was specific to PRAF2. 4. Conclusions We have characterized the gene structure and tissue distribution of human PRAF2, a new protein, which was recently found to interact with chemokine receptor CCR5. The PRAF2 topography model predicts a protein with four transmembrane domains with both amino- and carboxy-termini directed towards the cytoplasm. Sequence analyses also revealed a large PRA1 domain, an amphiphysin SH3 domain, and at least two putative phosphorylation sites within the PRAF2 amino acid sequence. The multiple sequence alignment with other PRA1 domain-containing proteins suggested that PRAF2 belongs to a new group of proteins, the PRAF protein family, which comprises the proteins PRAF1, PRAF2, and PRAF3. A phylogenetic analysis showed that these proteins are evolutionary conserved from yeast to man. PRAF2 is expressed in most human tissues and the results of a human tissue microarray revealed that the protein is overexpressed in tumor tissues of the breast, colon, lung, and ovary. Although the function of PRAF2 is not known, its close relation to PRAF1 and PRAF3 provides strong evidence that PRAF2 is a transport protein that predominantly localizes to the ER and functions in vesicular traffic in the cytoplasm. Since PRAF2 interacts with the cytoplasmic carboxy-terminus of CCR5 (Schweneker et al., 2005), it is possible that it acts as an adaptor protein during chemokine receptor internalization and recycling. This link may occur through the identified amphiphysin SH3 domain at the amino-terminus of PRAF2. This domain binds dynamin and forms a complex with β-arrestin, which plays a key role in clathrin-mediated receptor internalization. Furthermore, our recent findings in yeast suggest that PRAF2 associates with GDE1/MIR16 (Bachmann et al., 2006), a novel mammalian glycerophosphoinositol phosphodiesterase that interacts with the regulator of G protein signaling 16 (RGS16) (Zheng et al., 2000, 2003). Based on these observations, it is possible that the G protein-coupled receptor CCR5 forms a molecular protein complex with PRAF2, GDE1, and RGS16 that regulates CCR5, and possibly, other chemokine receptors (Bachmann et al., 2006). Finally, it will be of interest to see whether the functional diversity of PRAF2 is further expanded by the formation of homo-dimers as well as hetero-

dimers with PRAF1 and PRAF3 under physiological conditions. This report provides new information, which allows us to further investigate the biological role of PRAF2, and it introduces for the first time the new PRAF protein family. Acknowledgments We would like to thank Dr. Stuart Snyder, Andrew Hansen, and Mike Thorne for their technical assistance and helpful discussions. Dr. Carl-Wilhelm Vogel is thanked for his support during the course of this study. Dr. Brenda Hernandez and Hugh Luk are thanked for providing their expertise and services of the Histopathology Shared Resource (Cancer Research Center of Hawaii). We are also indebted to Invitrogen for the generous donation of four human tissue microarrays. This work was performed at the University of Hawaii and was funded by a career development grant from the Cancer Research Center of Hawaii to Dr. André S. Bachmann.

References Bachmann, A.S., Corpuz, G., Hareld, W.P., Wang, G., Coller, B.A., 2004. A simple method for the rapid purification of copia virus-like particles from Drosophila Schneider 2 cells. J. Virol. Methods 115, 159–165. Bachmann, A.S., Duennebier, F.F., Mocz, G., 2006. Genomic organization, characterization, and molecular 3D model of GDE1, a novel mammalian glycerophosphoinositol phosphodiesterase. Gene 371, 144–153. Blom, N., Gammeltoft, S., Brunak, S., 1999. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294, 1351–1362. Blom, N., Sicheritz-Ponten, T., Gupta, R., Gammeltoft, S., Brunak, S., 2004. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4, 1633–1649. Bucci, C., Chiariello, M., Lattero, D., Maiorano, M., Bruni, C.B., 1999. Interaction cloning and characterization of the cDNA encoding the human prenylated rab acceptor (PRA1). Biochem. Biophys. Res. Commun. 258, 657–662. Butchbach, M.E., Lai, L., Lin, C.L., 2002. Molecular cloning, gene structure, expression profile and functional characterization of the mouse glutamate transporter (EAAT3) interacting protein GTRAP3–18. Gene 292, 81–90. Cserzo, M., Wallin, E., Simon, I., von Heijne, G., Elofsson, A., 1997. Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: the dense alignment surface method. Protein Eng. 10, 673–676. Evergren, E., et al., 2004. Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse. Traffic 5, 514–528. Fan, Y., et al., 2004. Mutations in a member of the Ras superfamily of small GTP-binding proteins causes Bardet–Biedl syndrome. Nat. Genet. 36, 989–993. Horton, P., Nakai, K., 1997. Better prediction of protein cellular localization sites with the k nearest neighbors classifier. Proc. Int. Conf. Intell. Syst. Mol. Biol. 5, 147–152. Ingley, E., et al., 1999. A novel ADP-ribosylation like factor (ARL-6), interacts with the protein-conducting channel SEC61beta subunit. FEBS Lett. 459, 69–74. Inoue, K., Akiduki, S., Ikemoto, M.J., 2005. Expression profile of addicsin/ GTRAP3–18 mRNA in mouse brain. Neurosci. Lett. 386, 184–188. Kok, K., Naylor, S.L., Buys, C.H., 1997. Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. Adv. Cancer Res. 71, 27–92. Liang, Z., Li, G., 2000. Mouse prenylated Rab acceptor is a novel Golgi membrane protein. Biochem. Biophys. Res. Commun. 275, 509–516.

C.S. Fo et al. / Gene 371 (2006) 154–165 Liang, Z., Veeraprame, H., Bayan, N., Li, G., 2004. The C-terminus of prenylin is important in forming a dimer conformation necessary for endoplasmicreticulum-to-Golgi transport. Biochem. J. 380, 43–49. Lin, C.I., et al., 2001a. Modulation of the neuronal glutamate transporter EAAC1 by the interacting protein GTRAP3–18. Nature 410, 84–88. Lin, J., Liang, Z., Zhang, Z., Li, G., 2001b. Membrane topography and topogenesis of prenylated Rab acceptor (PRA1). J. Biol. Chem. 276, 41733–41741. Martincic, I., Peralta, M.E., Ngsee, J.K., 1997. Isolation and characterization of a dual prenylated Rab and VAMP2 receptor. J. Biol. Chem. 272, 26991–26998. Obenauer, J.C., Cantley, L.C., Yaffe, M.B., 2003. Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res. 31, 3635–3641. Owen, D.J., Wigge, P., Vallis, Y., Moore, J.D., Evans, P.R., McMahon, H.T., 1998. Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation. EMBO J. 17, 5273–5285. Schweneker, M., Bachmann, A.S., Moelling, K., 2005. JM4 is a fourtransmembrane protein binding to the CCR5 receptor. FEBS Lett. 579, 1751–1758. Seabra, M.C., Wasmeier, C., 2004. Controlling the location and activation of Rab GTPases. Curr. Opin. Cell Biol. 16, 451–457. Shaw, G., Kamen, R., 1986. A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659–667.

165

Sivars, U., Aivazian, D., Pfeffer, S.R., 2003. Yip3 catalyses the dissociation of endosomal Rab-GDI complexes. Nature 425, 856–859. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680. Wallick, C.J., et al., 2005. Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G1 cell cycle arrest in MYCNamplified human neuroblastoma cells. Oncogene 24, 5606–5618. Wigge, P., Vallis, Y., McMahon, H.T., 1997. Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain. Curr. Biol. 7, 554–560. Wykoff, C.C., et al., 2004. Gene array of VHL mutation and hypoxia shows novel hypoxia-induced genes and that cyclin D1 is a VHL target gene. Br. J. Cancer 90, 1235–1243. Zheng, B., Chen, D., Farquhar, M.G., 2000. MIR16, a putative membrane glycerophosphodiester phosphodiesterase, interacts with RGS16. Proc. Natl. Acad. Sci. U. S. A. 97, 3999–4004. Zheng, B., Berrie, C.P., Corda, D., Farquhar, M.G., 2003. GDE1/MIR16 is a glycerophosphoinositol phosphodiesterase regulated by stimulation of G protein-coupled receptors. Proc. Natl. Acad. Sci. U. S. A. 100, 1745–1750.