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• W2000011561 abstract "Murine Pactolus is a β-integrin-like molecule expressed exclusively on the surface of granulocytes. Cell surface expression of Pactolus is dramatically increased following activation of bone marrow neutrophils with known agonists, and cross-linking of cell surface Pactolus, suggesting the bulk of the protein is in intracellular stores. The mature protein is found in two forms depending upon the extent of N-linked glycosylation. There is no evidence to suggest that Pactolus requires an associated α chain for expression. In some mouse strains, a truncated form of the protein is predicted based upon alternative splicing: this form, however, is unstable and rapidly degraded after synthesis. Differences in the quantities of these Pactolus mRNA isoforms have defined two alleles. BALB/c and C3H/HeJ mice possess allele B and preferentially express the truncated, unstable product, whereas C57BL/6 mice possess allele A and only produce the membrane-bound form. Sequence analysis has shown the difference between these two alleles is due to a single base pair difference at the splice acceptor site for the truncated product. The increased expression of the membrane form of Pactolus by granulocytes of C57BL/6 mice suggests a compensatory adhesion function that is reduced in cells from the low producing strains. Murine Pactolus is a β-integrin-like molecule expressed exclusively on the surface of granulocytes. Cell surface expression of Pactolus is dramatically increased following activation of bone marrow neutrophils with known agonists, and cross-linking of cell surface Pactolus, suggesting the bulk of the protein is in intracellular stores. The mature protein is found in two forms depending upon the extent of N-linked glycosylation. There is no evidence to suggest that Pactolus requires an associated α chain for expression. In some mouse strains, a truncated form of the protein is predicted based upon alternative splicing: this form, however, is unstable and rapidly degraded after synthesis. Differences in the quantities of these Pactolus mRNA isoforms have defined two alleles. BALB/c and C3H/HeJ mice possess allele B and preferentially express the truncated, unstable product, whereas C57BL/6 mice possess allele A and only produce the membrane-bound form. Sequence analysis has shown the difference between these two alleles is due to a single base pair difference at the splice acceptor site for the truncated product. The increased expression of the membrane form of Pactolus by granulocytes of C57BL/6 mice suggests a compensatory adhesion function that is reduced in cells from the low producing strains. metal ion-dependent adhesion site phorbol myristate acetate polymerase chain reaction fluorescence-activated cell sorter phosphate-buffered saline horseradish peroxidase endoglycosidase H fluorescein isothiocyanate polyacrylamide gel electrophoresis phycoerythrin horseradish peroxidase radioimmune precipitation buffer Pactolus coding sequences were first isolated in a differential display screen of mouse marrow differentiated into cell subtypes in the presence of stem cell factor or interleukin-3 (1Chen Y. Garrison S. Weis J.J. Weis J.H. J. Biol. Chem. 1998; 273: 8711-8718Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Pactolus was expressed in those cells maintained in stem cell factor, but lost when cells were grown in interleukin-3. In the mature animal, the tissue of highest expression was the bone marrow. The Pactolus gene encodes, via alternative splicing, two distinct isoforms of products that share homology with the β-integrins, primarily β2 and β7. The full-length form, dubbed Pac A, resides on the membrane as a class I type glycoprotein (1Chen Y. Garrison S. Weis J.J. Weis J.H. J. Biol. Chem. 1998; 273: 8711-8718Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The truncated form, Pac B, lacks transmembrane and cytoplasmic sequences and, if stable, is predicted to be a soluble and/or secreted form. These differing transcripts are due to an alternative splicing event within exon 13 of the gene. A third form of Pactolus transcript, Pac C, which also predicts a truncated protein, is also due to alternative splicing near this exon; however, it represents only a minor constituent of Pactolus transcripts in the mature animal (2Margraf R.L. Chen Y. Garrison S. Weis J.J. Weis J.H. Mamm. Genome. 1999; 10: 1075-1081Crossref PubMed Scopus (7) Google Scholar). Pactolus has been referred to as β-integrin-like due to the lack of conservation within the metal ion-dependent adhesion site (MIDAS)1 domain that is maintained in the integrin subunits (3Humphries M.J. Curr. Opin. Cell Biol. 1996; 8: 632-640Crossref PubMed Scopus (203) Google Scholar). Pactolus has a point mutation in the critical DXSXS motif required for a functional MIDAS domain and has an apparent deletion of 28 amino acids immediately carboxyl-terminal to this site (1Chen Y. Garrison S. Weis J.J. Weis J.H. J. Biol. Chem. 1998; 273: 8711-8718Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Mutations within the MIDAS domains of other integrin subunits abrogates heterodimer formation and ligand recognition (4Bajt M.L. Loftus J.C. J. Biol. Chem. 1994; 269: 20913-20919Abstract Full Text PDF PubMed Google Scholar, 5Wardlaw A.J. Hibbs M.L. Stacker S.A. Springer T.A. J. Exp. Med. 1990; 172: 335-345Crossref PubMed Scopus (81) Google Scholar, 6Michishita M. Videm V. Arnaout M.A. Cell. 1993; 72: 857-867Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 7Ueda T. Rieu P. Brayer J. Arnaout M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10680-10684Crossref PubMed Scopus (162) Google Scholar, 8Rieu P. Ueda T. Haruta I. Sharma C.P. Arnaout M.A. J. Cell Biol. 1994; 127: 2081-2091Crossref PubMed Scopus (99) Google Scholar). Immunoprecipitation analyses of Pactolus have not identified an α partner chain even when such experiments were performed under conditions that maintained other α/β-integrin pairs (1Chen Y. Garrison S. Weis J.J. Weis J.H. J. Biol. Chem. 1998; 273: 8711-8718Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The high degree of sequence homology between Pactolus and the β-integrins is evident in the extracellular domains; however, the cytoplasmic region of the Pactolus protein does not possess similar sequences (9Wilson R.W. O'Brien W.E. Beaudet A.L. Nucleic Acids Res. 1989; 17: 5397Crossref PubMed Scopus (30) Google Scholar). Exon/intron mapping of the Pactolus gene suggests Pactolus and β2-integrin arose from a common precursor even though they now reside on different mouse chromosomes (2Margraf R.L. Chen Y. Garrison S. Weis J.J. Weis J.H. Mamm. Genome. 1999; 10: 1075-1081Crossref PubMed Scopus (7) Google Scholar). In this article we report our investigations pertaining to the genetics and biology of the production of the truncated and full-length Pactolus products. Using a variety of anti-Pactolus antisera, we demonstrate that the maturing and mature neutrophil is the major cell type that expresses Pactolus. We show that the full-length Pactolus product is stably expressed on the cell surface and undergoes extensiveN-linked glycosylation, whereas the truncated Pactolus product is unstable and rapidly degraded immediately after synthesis. Cross-linking of cell surface Pactolus leads to neutrophil activation and the rapid mobilization of intracellular stores of Pactolus to the cell surface. Finally, a number of mouse strains have been analyzed and two distinct Pactolus alleles have been found. These alleles appear to be directly responsible for the quantities of Pactolus transcript (and protein) isoforms found in selected strains; these differences are due to single nucleotide alteration in a splice acceptor site of the truncated product. Inbred mice (BALB/c, C57BL/6, C3H/HeJ) and outbred mice (Swiss NIH) were obtained from NCI, National Institutes of Health (Bethesda, MD). Tissues were isolated from adults (greater than 10 weeks of age) or immature animals (10 days). Bone marrow cells were obtained by flushing femurs with RPMI without fetal calf serum. Activation of purified bone marrow cells was done using 20 ng/ml PMA for FACS analysis and 100 ng/ml PMA for degranulation assays. Cells were activated with the Bac Pac antiserum at a 1:500 dilution of that serum (or the pre-immune control). The extracellular Pactolus coding sequence (minus the Pactolus signal sequence and missing those sequences encoding the transmembrane and cytoplasmic domains) was PCR-amplified with Pfu polymerase (Stratagene, San Diego, CA) and inserted within the Baculovirus cloning vector pAcGP67B at the EcoRI site (PharMingen, San Diego, CA). This vector supplies the signal sequence for membrane insertion and secretion into the supernatant, and adds the 6-His tag sequence to the carboxyl terminus of the protein. Sf9 cells were transfected with the Pactolus expression plasmid and BaculoGold, the linear recombination substrate to generate a stable line of cells producing the recombinant baculovirus. Virus supernatants were used to infect 1.8 × 107 cells. Supernatants were collected 6 days after infection. After overnight dialysis against PBS, pH 8.0, the supernatants were brought up to 20 mm imidazole. They were then incubated for 60 min with 2 ml of nickel-nitrilotriacetic acid slurry (Qiagen). After the incubation, the mixture was bound to a column, washed two times with 10 ml of wash solution (1× PBS, pH 8.0, and 20 mm imidazole), and protein eluted in 1-ml aliquots using elution buffer (1× PBS, pH 8.0, and 500 mmimidazole). Two rabbits were injected with the purified Bac Pac protein (5 μg/animal). Although both animals responded to the immunization, the highest titer antiserum from one of the animals was used for experimentation purposes. The antiserum was defined to be specific for Pactolus based upon its FACS, Western blot, and immunoprecipitation analyses (detailed in the present report). Lewis rats were immunized with the Pactolus recombinant protein. Rat sera were tested until Pactolus-specific antibodies were detected. Splenic cells were disassociated from the spleen, washed, and counted. Cells (2.8 × 107) were incubated with equal amounts of SP2/0 cells. Splenocytes and SP2/0 cells were fused over 1 min using 30% polyethylene glycol. Cells were then centrifuged and plated in complete media (RPMI 1640, 10% penicillin/streptomycin, 10%l-glutamine, 10% nonessential amino acids, 10% fetal calf serum, and 20 μg/ml insulin) over 10 96-well plates. 24 h after fusion, cells were incubated in complete media with 1× HAT. Cells were fed every 3 days. The supernatants from every well were then screened for a Pactolus-specific antibody by enzyme-linked immunosorbent assay with the His-tagged Bac Pac protein. Positive wells were diluted down serially to obtain a single cell in a single well. The B10 anti-Pactolus monoclonal antibody recognizes the Pactolus protein in denaturing (RIPA solution; see below) immunoprecipitations but does not recognize the protein via FACS or Western blot analyses. RNA transcription was performed with an RNA transcription kit (Stratagene, La Jolla, CA). Briefly, 1 μg of template DNA was incubated with 5 μl of 5× transcription buffer, 4 μl of 10 mm rNTP, 1 μl of 0.75m dithiothreitol, 10 units of T7 RNA polymerase, and water to a final volume of 25 μl. The mixture was incubated at 37 °C for 30 min. The RNA was phenol/chloroform-extracted and precipitated in ethanol. The translation of the purified RNA was performed with a rabbit reticulocyte lysate system (Promega, Madison, WI). Briefly, 2 μg of template RNA was incubated with 35 μl of rabbit reticulocyte lysate, 1 μl of 1 mm methionine minus amino acid mix, 15 mCi/ml [35S]methionine, and water to a final volume of 50 μl. The mixture was incubated at 30 °C for 90 min. Samples were immunoprecipitated and visualized as described under immunoprecipitation. Total RNA was isolated using CsCl guanidine (10Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16654) Google Scholar). RNA was resuspended in water and quantified byA 260 absorbance. cDNA was synthesized from 5 μg of RNA, 10 μl of 5× first strand buffer, 5 μl of 5 mm dNTP, 5 μl of 0.1 m dithiothreitol, 2 μl of 1.25 mm random primers (Life Technologies, Inc.), 2 μl of Moloney murine leukemia virus reverse transcriptase, and water to final volume of 50 μl. The reaction was incubated at 37 °C for 1 h. 2 μl of DNase-free RNase (1 mg/ml) was added, and the reaction incubated for an additional 5 min followed by cDNA isolation via ethanol precipitation. PCR using radioactive detection was performed with 10-μl reactions containing 200 ng of cDNA, 70 pm amounts of each primer, 0.72 unit of Ampli-Taq DNA polymerase (Life Technologies, Inc.), 0.8 mm dNTP, 1× Taq buffer (50 mm TriS, pH 8.3, 3 mm MgCl2, 20 mm KCl, and 500 mg/ml bovine serum albumin), and 2.5 μCi of [32P]dCTP. Samples were loaded into capillary tubes and incubated in an air thermocycler (Idaho Technology, Idaho Falls, ID) for denaturing at 94 °C for 1 s, for annealing at 60 °C for 1 s, and for extension at 72 °C for 3 s (11Tan S.S. Weis J.H. Genome Res. 1992; 2: 137-143Crossref Scopus (61) Google Scholar). This cycle was repeated 14 times for amplification of β-actin transcripts and 23 times for amplification of Pactolus transcripts. After amplification, 10 μl of stop solution (U. S. Biochemical Corp) was added, and 5 μl was resolved in a 6% acrylamide sequencing gel for autoradiography. Gene-specific oligonucleotides (which span an intron) were: β-actin, 62GTA ACA ATG CCA TGT TCA AT and339CTC CAT CGT GGG CCG CTC TAG; Pactolus,827TGT ACT TCC TGA TGG GCC TC, 828TGT ACT TCC TGA TGG GCC TC, 837CGA GTG CGA CAA TGT CAA CTG, and848TAG TAC TCG GAG CAG CGA TGG. Bone marrow cells were harvested from mice, and red blood cells were lysed with red blood cell lysis solution (0.15 m NH4Cl, 1.0 mmKHCO3, and 0.1 mm EDTA, pH 7.2) for 4 min at 4 °C. Cells were then washed in methionine- and serum-free RPMI two times, incubated for 1 h in methionine- and serum-free RPMI, then labeled for 1 h with 0.5 mCi/ml [35S]methionine. Chase after labeling was for 1–6 h with normal serum/media. Cells were then centrifuged and lysed in RIPA solution (50 mm NaCl, 25 mm Tris, pH 7.5, 1 mm EDTA, 0.1% SDS, 1% sodium deoxycholate, 1% Nonidet P-40, 1% bovine serum albumin, 1 mm phenylmethylsulfonyl fluoride, and complete TM (Mini protease inhibitor mixture tablet; Roche Molecular Biochemicals) for 12 min. Cell lysates were collected and precleared by incubating with pre-immune rabbit serum and 10 mg of protein A-Sepharose overnight at 4 °C. The lysates were then precleared two times with 12.5 mg of protein G-Sepharose to remove native labeled IgG. The supernatants were then incubated with either polyclonal rabbit antiserum against the Bac Pac extracellular protein or the cytoplasmic tail sequence. The protein antibody complex was then absorbed with protein A-Sepharose for 30 min at 4 °C. After absorption the Sepharose beads were washed five times: one time with 1 m NaCl and RIPA, two times with RIPA buffer, and twice with a final wash solution (50 mmNaCl, 25 mm Tris, pH 7.5, and 1 mm EDTA). The samples were then boiled under reducing (+ dithiothreitol) or non-reducing (− dithiothreitol) 2× sample buffer (4× Tris/SDS, pH 6.8, 20% glycerol, 4% SDS, ± 0.2 m dithiothreitol, 0.012% bromphenol blue) for 5 min before they were loaded on to an 8% Tris-glycine polyacrylamide gel. After electrophoresis the gel was treated with Coomassie stain for 15 min, destain solution (40% methanol and 10% acetic acid) for 15 min, and Amplify solution (Amersham Life Sciences) for 20 min. Gel was then dried and placed overnight on Kodak X-Omat-AR film. Deglycosylation enzymes were purchased from Glyko (Novato, CA) unless otherwise stated: NANase III, specific for α2–3,6,8,9 N-acetylneuraminic acid; sialadase, specific for α2–3,6,8 N-acetylneuraminic acid;N-glycanase, specific for β-aspartyl-glycosylamine bond at asparagine residue; and endoglycosidase H (Endo H; Roche Molecular Biochemicals), cleavage between the GlcNAc residues of the chitobiose unit of N-glycans linked to asparagine. The glycoproteins were first immunoprecipitated with the B10 Pactolus monoclonal antibody, and the precipitates were incubated with the appropriate enzyme using standard protocols and buffers supplied by Glyko. Endo H cleavage was performed by first placing the pellet in 0.1m 2-mercaptoethanol, 0.1% SDS and then heat-denaturing for 5 min at 90 °C. Sample was centrifuged, and the supernatant was removed and incubated in cleavage buffer (75 mm sodium citrate, pH 5.5, 0.05% phenylmethylsulfonyl fluoride, 0.5 unit/ml Endo H) overnight at 37 °C. SDS-PAGE was then performed on all samples followed by Western analysis. SDS-PAGE was performed on immunopreciptitation samples under reducing conditions. After electrophoresis PVDF membrane (polyvinylidene fluoride, Immobilon-P, Millipore) was prepared following the company's protocol, and the proteins were transferred for 1.25 h at 30 V. After transfer, the membrane was soaked in 100% methanol for 20 s and then dried for 20 min. Membrane was then placed in blocking solution (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 5% milk) for 15 min and incubated with 1:1000 dilution of the anti-Pactolus Bac Pac polyclonal antibody for 1 h. Membrane was then washed twice (10 min each wash) in Tris-buffered saline-Tween (20 mmTris-HCl, pH 7.5, 150 mm NaCl, 0.05% Tween 20). Membrane was then placed in blocking buffer containing 1:5000 dilution of goat anti-rabbit HRP (Bio-Rad) for 30 min. Membranes were again washed twice (10 min each wash) in Tris-buffered saline-Tween and then incubated for 5 min with SuperSignal® chemiluminescent substrate (Pierce) and visualized on blue x-ray film. The Western blot performed using avidin-HRP used blocking solution consisting of PBS with 1% bovine serum albumin followed by washes in PBS-Tween. Mouse bone marrow cells were obtained from adult or immature animals. Red blood cells were lysed as described previously. 2 × 106 cells were used for each staining reaction. Peritoneal neutrophils were obtained by injection of 1 ml of a 1% solution of oyster glycogen into the peritoneal cavity of mice, followed by aspiration of the cavity 16 h later. Cells were collected, red blood cells lysed, and the resulting cells analyzed by FACS. The rabbit anti-Bac Pac was detected using a FITC-conjugated goat anti-rabbit Ig antibody (Cappel). Antibodies specific for Gr-1(clone RB6–8C5), B220 (clone RA3–6B2), and Mac-1 (clone M1/70) were obtained from the University of Utah Stem Cell core facility and were directly conjugated with phycoerythrin (PE). We previously documented the expression of the full-length, transmembrane form of Pactolus on adult bone marrow cells using antisera generated against the cytoplasmic region (anti-CT) (1Chen Y. Garrison S. Weis J.J. Weis J.H. J. Biol. Chem. 1998; 273: 8711-8718Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). To generate Pactolus-specific antisera, the extracellular portion of the full-length form (excluding the signal sequence, transmembrane, and cytoplasmic domains of Pactolus) was inserted into the Baculovirus vector pAcGP67B. The Baculovirus-derived Pactolus protein (termed Bac Pac) was used to generate a polyclonal rabbit antiserum (Bac Pac antiserum) (Fig.1 A). This antiserum can detect the Pactolus gene product by immunoprecipitation, Western blot, and FACS analysis (see below). The Baculovirus-derived Pactolus protein was also used to create a monoclonal antibody (see below). The anti-Pactolus Bac Pac polyclonal antiserum was tested for recognition of the Pactolus protein using in vitrotranscription and translation. The open reading frame of the Pac A transcript was placed in a T7 promoter expression plasmid. The coding sequence was truncated by digestion of the plasmid with single cutting enzymes SmaI (which would be predicted to encode a truncated protein of M r 62,000) and HpaI (which would be expected to encode a truncated protein ofM r 41,000) (Fig. 1 B). The full-length Pactolus peptide sequence encodes a protein ofM r 81,000. Translation and immunoprecipitation of these truncated proteins indicated that the polyclonal Bac Pac antiserum recognizes epitopes present in the HpaI- andSmaI-generated products. Therefore the Bac Pac antiserum should be functional for the detection of both the Pac A (full-length) and Pac B (truncated) protein products. The Bac Pac antiserum was then tested for its ability to recognize Pactolus expressed on the surface of cells. Previously we had determined bone marrow samples expressed the highest level of Pactolus transcripts (1Chen Y. Garrison S. Weis J.J. Weis J.H. J. Biol. Chem. 1998; 273: 8711-8718Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Therefore, we obtained bone marrow from 10-day-old and 10-week-old C57BL/6, C3H/HeJ, and BALB/c mice and analyzed by FACS using Bac Pac antiserum with pre-immune serum as control. As shown in Fig. 2 A, the Bac Pac antiserum specifically stained ∼50% of the bone marrow cells from the adult and juvenile C57BL/6 mouse (M1) marrow. The marrow from the C3H/HeJ and BALB/c mice demonstrated a slight positive shift regardless of age. The depressed levels of Pactolus protein on the surface of the BALB/c and C3H/HeJ bone marrow cells, compared with the C57BL/6 cells, suggested either 1) there are fewer Pactolus transcripts in the C3H/HeJ and BALB/c cells or 2) the production of the spliced isoform of Pactolus to produce the full-length, transmembrane form of the protein is decreased in the BALB/c and C3H/HeJ cells compared with those of the C57BL/6 animal. cDNA was prepared from RNA isolated from total bone marrow cells obtained from these mice and analyzed, via semiquantitative reverse transcriptase-PCR, for either total Pactolus transcripts or those forms that, through alternative splicing, produce either the full-length, transmembrane protein or the truncated form. For the former assessment oligonucleotides were used that span a region of the gene present in all Pactolus transcripts, and for the latter oligonucleotides that span the alternatively spliced junction were used. The C57BL/6 sample appeared to possess only slightly more total Pactolus transcripts than the BALB/c samples (Fig. 2 B). In the C57BL/6 sample, the only Pactolus transcript was that encoding the transmembrane, Pac A form whereas the BALB/c cells were highly enriched for transcripts encoding the Pac B, truncated form. Since the truncated form of Pactolus does not encode a transmembrane-spanning region for cell surface localization, this protein must be degraded, stored within the cell, or secreted (see below). This difference in transcript profile between the C57BL/6 and the other strains could easily account for the difference in cell surface staining intensity for Pactolus. Major populations of cells in the marrow include B cell and granulocyte lineages that can be distinguished with monoclonal antibodies specific for B220 and Gr-1, respectively (12Hathcock K.S. Hirano H. Murakami S. Hodes R.J. J. Immunol. 1992; 149: 2286-2294PubMed Google Scholar, 13Hestdal K. Ruscetti F.W. Ihle J.N. Jacobsen S.E. Dubois C.M. Kopp W.C. Longo D.L. Keller J.R. J. Immunol. 1991; 147: 22-28PubMed Google Scholar). In order to determine the lineage of cells that express Pactolus, bone marrow cells from C57BL/6, C3H/HeJ, and BALB/c mice were analyzed by two-color FACS analysis with anti-Gr-1, anti-B220, and anti-Mac-1 (14Springer T. Galfre G. Secher D.S. Milstein C. Eur. J. Immunol. 1979; 9: 301-306Crossref PubMed Scopus (870) Google Scholar). Shown in Fig.3 A, cells from the C57BL/6 strain that expressed Pactolus also possessed the Gr-1 marker and did not express B220. Pactolus-positive cells also did not stain with CD19 (15Tedder T.F. Zhou L.J. Engel P. Immunol. Today. 1994; 15: 437-442Abstract Full Text PDF PubMed Scopus (240) Google Scholar) (data not shown). These results indicated that cells of the granulocytic, but not lymphocytic, lineage express Pactolus. Mac-1 expression indicated that there is a subset of cells within the marrow that stain brightly for both Mac-1 and Pactolus, another that only stains for Mac-1, and a third subset that is not recognized by either antibody. These data also demonstrate that the Bac Pac antiserum does not cross react with the β2-integrin subunit, which is part of the Mac-1 complex. The analysis of bone marrow cells from the C3H/HeJ and BALB/c strains demonstrated only slight staining of Pactolus on the Gr-1 subset, as expected from the transcript data described above. Bone marrow was further analyzed by FACS Vantage cell sorting using the Bac Pac antiserum to fractionate the marrow (Fig. 3 B). Two populations (positive and negative for Pactolus staining) were collected, spun onto glass coverslips, and analyzed by Wright stain. The cells found within the negative population were heterogeneous but included a large percentage of immature B lymphocytes. The majority of cells in this population stained with B220 (see Fig. 3 A). Alternatively, the Pactolus-positive subset was highly enriched for cells of the neutrophil lineage. Cells representing each stage of neutrophil development were apparent in this stain. Gr-1-positive cells in the bone marrow express Pactolus. A key question was whether Pactolus is preferentially expressed in the bone marrow or if Gr-1-positive cells (primarily neutrophils) still express Pactolus once they enter the periphery of the animal. The anti-Pactolus Bac Pac antiserum was used with two-color staining, using anti-Gr-1, to analyze total splenocytes from C57BL/6 animals. As shown in Fig.4 A, only a small percentage of cells in the spleen stain brightly with Gr-1 (R2). Of these, virtually all possess Pactolus (R3). Again, these data demonstrate that the Bac Pac antiserum does not react with the β2-integrin, which is expressed on splenic T and B cells. To test for Pactolus expression by mature neutrophils in the periphery, oyster glycogen was injected into the peritoneal cavity to recruit neutrophils (16Mulligan M.S. Lentsch A.B. Ward P.A. Inflammation. 1998; 22: 327-339Crossref PubMed Scopus (11) Google Scholar). After 16 h, aspirated cells from the peritoneal cavity are highly enriched for neutrophils. We performed such a strategy using C57BL/6, BALB/c, and C3H/HeJ mice and analyzed the resulting cells for the co-expression of Pactolus and Gr-1 (Fig.4 B). In the C57BL/6 sample, all of the peritoneal cells that stained for Gr-1 also expressed Pactolus. As expected, the same subset of Gr-1-positive cells in the BALB/c and C3H/HeJ stained only minimally for Pactolus. These data indicate that the primary cell of the mouse that expresses Pactolus is the maturing and mature neutrophil, and that the mouse strain differences in Pactolus cell surface expression identified for the bone marrow granulocytes were recapitulated in peripheral neutrophils. The Pactolus gene encodes two primary transcripts that utilize alternative splicing at exon 13 to generate the full-length form, Pac A, or a truncated form, Pac B (1Chen Y. Garrison S. Weis J.J. Weis J.H. J. Biol. Chem. 1998; 273: 8711-8718Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 2Margraf R.L. Chen Y. Garrison S. Weis J.J. Weis J.H. Mamm. Genome. 1999; 10: 1075-1081Crossref PubMed Scopus (7) Google Scholar). The Pac B transcript predicts a primary protein ofM r 55,000, whereas the full-length form predicts a primary protein of M r 81,000. The reactivity of the Bac Pac antiserum with the in vitrotranscription/translation products suggests it should detect both of these Pactolus products. Mouse bone marrow cells from C57BL/6 mice were labeled with [35S]Met and chased with cold methionine for different periods of time. Samples were immunoprecipitated with pre-immune serum, the Bac Pac antiserum, and the antiserum raised against the Pactolus cytoplasmic tail sequence (1Chen Y. Garrison S. Weis J.J. Weis J.H. J. Biol. Chem. 1998; 273: 8711-8718Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Both the cell pellet and the supernatant were analyzed for Pactolus protein products (Fig. 5 A). At the 0 chase time (left panel), the M r98,000 mature Pactolus product was observed with the Bac Pac and cytoplasmic region antisera. After the 6-h label, a second diffuse band of M r ∼130,000 was evident in addition to theM r 98,000 protein, suggesting that the smaller protein had been further modified during the time course of the labeling. We have never been able to fully “chase” theM r 98,000 form into theM r 130,00 form, suggesting that a portion of the Pactolus product does not bear this increased modification. TheM r 98,000 form is also the size of the full-length Pactolus protein expressed on the surface of Chinese hamster ovary cells transfected with an expression construct encoding the full-length Pactolus product (data not shown). The same type of analysis was carried out with bone marrow cells obtained from BALB/c mice. Cells were labeled with [35S]Met and chased with cold methionine for 0 and 6 h. Lysates and cell supernatants were immunoprecipitated with the Bac Pac and pre-immune antisera. As shown in Fig. 5 B, neither the full-length or truncated Pactolus product was evident. Less than 10% of the Pactolus transcripts from the BALB/c cells encode the full-length Pac A product and since we can only barely detect Pactolus expression on the surface of BALB/c bone marrow neutrophils by FACS analysis, it is not surprising that we do not readily observe theM r 98,000 protein. However, the truncated Pac B product should be evident in either the supernatant as a secreted product or within a storage compartment of the BALB/c cells. Since the level of total Pactolus transcripts between t" @default.
• W2000011561 created "2016-06-24" @default.
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• W2000011561 date "2001-09-01" @default.
• W2000011561 modified "2023-09-27" @default.
• W2000011561 title "Functional Characterization of Pactolus, a β-Integrin-like Protein Preferentially Expressed by Neutrophils" @default.
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