Murine HIP/L29 Is a Heparin-binding Protein with a Restricted Pattern of Expression in Adult Tissues
1998; Elsevier BV; Volume: 273; Issue: 39 Linguagem: Inglês
10.1074/jbc.273.39.25148
ISSN1083-351X
AutoresDavid E. Hoke, E. Gloria C. Regisford, JoAnne Julian, Asna Amin, Catherine Bègue‐Kirn, Daniel D. Carson,
Tópico(s)Peptidase Inhibition and Analysis
ResumoHeparin/heparan sulfate (Hp/HS)-binding proteins are implicated in a variety of cell biological processes including cell adhesion, modulation of blood coagulation, and cytokine/growth factor action. Hp/HS-interacting protein (HIP) has been identified in various adult tissues in humans. HIP supports high affinity, selective binding to Hp/HS, promotes cell adhesion, and modulates blood coagulation activities via Hp/HS-dependent mechanisms. Herein, a murine ortholog of human HIP is described that is 78.8% identical to human HIP and 99.8% identical at the cDNA level and identical at the amino acid level to a previously described murine ribosomal protein, L29. Western blot analyses and immunohistological staining with affinity-purified antibodies generated against two distinct peptide sequences of murine HIP/L29 indicate that HIP/L29 is differentially expressed in adult murine tissues and cell types. In the normal murine mammary epithelial cell line, NMuMG, HIP/L29 is enriched in the 100,000 × g particulate fraction. HIP/L29 can be solubilized from the 100,000 × g particulate fraction with 0.8 m NaCl, suggesting that it is a peripheral membrane protein. HIP/L29 directly binds 125I-Hp in gel overlay assays and requires 0.75 m NaCl for elution from Hp-agarose. In addition, recombinant murine HIP expressed in Escherichia coli binds Hp in a saturable and highly selective manner, compared with other glycosaminoglycans including dermatan sulfate, chondroitin sulfate, keratan sulfate, and hyaluronic acid. Collectively, these data indicate that murine HIP/L29, like its human ortholog, is a Hp-binding protein expressed in a restricted manner in adult tissues. Heparin/heparan sulfate (Hp/HS)-binding proteins are implicated in a variety of cell biological processes including cell adhesion, modulation of blood coagulation, and cytokine/growth factor action. Hp/HS-interacting protein (HIP) has been identified in various adult tissues in humans. HIP supports high affinity, selective binding to Hp/HS, promotes cell adhesion, and modulates blood coagulation activities via Hp/HS-dependent mechanisms. Herein, a murine ortholog of human HIP is described that is 78.8% identical to human HIP and 99.8% identical at the cDNA level and identical at the amino acid level to a previously described murine ribosomal protein, L29. Western blot analyses and immunohistological staining with affinity-purified antibodies generated against two distinct peptide sequences of murine HIP/L29 indicate that HIP/L29 is differentially expressed in adult murine tissues and cell types. In the normal murine mammary epithelial cell line, NMuMG, HIP/L29 is enriched in the 100,000 × g particulate fraction. HIP/L29 can be solubilized from the 100,000 × g particulate fraction with 0.8 m NaCl, suggesting that it is a peripheral membrane protein. HIP/L29 directly binds 125I-Hp in gel overlay assays and requires 0.75 m NaCl for elution from Hp-agarose. In addition, recombinant murine HIP expressed in Escherichia coli binds Hp in a saturable and highly selective manner, compared with other glycosaminoglycans including dermatan sulfate, chondroitin sulfate, keratan sulfate, and hyaluronic acid. Collectively, these data indicate that murine HIP/L29, like its human ortholog, is a Hp-binding protein expressed in a restricted manner in adult tissues. heparin heparan sulfate bovine serum albumin phosphate-buffered saline polyacrylamide gel electrophoresis. Heparin/heparan sulfate (Hp/HS)1 proteoglycans and their respective binding proteins play important roles in diverse biological processes including cell adhesion, cytokine/growth factor action, and modulation of blood coagulation (1Salmvirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (398) Google Scholar, 2Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar, 3Lindahl U. Höök M. Annu. Rev. Biochem. 1978; 47: 385-417Crossref PubMed Scopus (586) Google Scholar, 4Middleton J. Stuart N. Wintle J. Clark-Lewis I. Moore H. Lam C. Auer M. Hub E. Rot A. Cell. 1997; 91: 385-395Abstract Full Text Full Text PDF PubMed Scopus (633) Google Scholar). Furthermore, several lines of evidence indicate that HS proteoglycans and their binding proteins participate in aspects of embryo implantation (5Carson D.D. Rohde L.H. Surveyor G. Int. J. Biochem. 1994; 26: 1269-1277Crossref PubMed Scopus (27) Google Scholar). Recently, human Hp/HS-interacting protein (HIP) has been identified in uterine endometrium with expression in lumenal uterine epithelial cells, i.e. the cells to which embryos initially attach during the implantation process (6Rohde L.H. Julian J. Babaknia A. Carson D.D. J. Biol. Chem. 1996; 271: 11824-11830Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The expression pattern of HIP coupled with its function as a Hp/HS-binding protein makes it a candidate protein involved in Hp/HS-dependent embryo adhesion. Previous reports from our laboratory have characterized human HIP as a cell surface, Hp/HS-binding protein expressed in a number of normal human tissues and cell lines (6Rohde L.H. Julian J. Babaknia A. Carson D.D. J. Biol. Chem. 1996; 271: 11824-11830Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 7Liu S. Smith S.E. Julian J. Rohde L.H. Karin N.J. Carson D.D. J. Biol. Chem. 1996; 271: 11817-11823Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). HIP mRNA is 1.3 kilobases in length and encodes a protein with a calculated molecular mass of 17,750 kDa; however, HIP migrates at an apparent molecular mass of 24 kDa by SDS-PAGE, apparently due to its very high isoelectric point. HIP mRNA was found in a variety of epithelial cell types, but was absent or at markedly reduced levels in two fibroblastic cell lines examined. HIP protein expression was examined immunohistochemically in human uterine endometrial sections where it was localized to the uterine and glandular epithelium and also on the vascular endothelium. Purified human HIP as well as a recombinant HIP protein expressed in Escherichia coli were shown to bind Hp selectively and saturably and also support Hp/HS-dependent cell adhesion (8Liu S. Hoke D. Julian J. Carson D.D. J. Biol. Chem. 1997; 272: 25856-25862Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In addition to reports on HIP protein, a peptide sequence within HIP has been shown to bind specific Hp/HS sequences within the Hp/HS chain (9Liu S. Julian J. Carson D.D. J. Biol. Chem. 1998; 273: 9718-9762Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Notably, this peptide domain binds the same Hp/HS pentasaccharide that is recognized by the anticoagulant protein, antithrombin III (10Liu S. Zhou F. Höök M. Carson D.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1739-1744Crossref PubMed Scopus (64) Google Scholar). Consequently, HIP is thought to compete with antithrombin III for this pentasaccharide motif and modulate blood coagulation activities in vivo. This present study describes the cDNA cloning, expression, and function of a murine ortholog to human HIP that is 99.8% identical to a previously published cDNA sequence for mouse ribosomal protein L29 (11Rudert F. Garnier J. Schuhbaur B. Gene (Amst.). 1993; 133: 249-254Crossref PubMed Scopus (7) Google Scholar). Using two antibodies generated toward distinct peptide sequences of this protein, the expression pattern of murine HIP/L29 was investigated. Additionally, recombinant murine HIP/L29 expressed in E. coli retains the ability to bind Hp selectively and saturably. Like human HIP, murine HIP/L29 is a protein that binds Hp with a high degree of selectivity and is expressed in a cell type-restricted fashion in adult mouse tissues. Heparin, dermatan sulfate, chondroitin sulfate C, keratan sulfate, hyaluronic acid, sodium chloride, sodium citrate, Tris base, glycine, bovine serum albumin (BSA), and phenylmethylsulfonyl fluoride were purchased from Sigma. [3H]Hp (0.44 mCi/mg) was purchased from NEN Life Science Products. 125I-Protein A (45 mCi/mg) was purchased from ICN Biochemicals Inc. (Irvine, CA). Sodium dodecyl sulfate (SDS), β-mercaptoethanol, and Tween 20 were purchased from Bio-Rad. All chemicals used were reagent grade or better. A mouse uterine cDNA library constructed in the λ ACT vector (provided by Dr. Joe Miano, M. D. Anderson Cancer Center) was used for cDNA library screening. Nitrocellulose filters (Schleicher & Schuell) lifted off the plated cDNA library were prehybridized at high stringency (50% (v/v) formamide, 2× SSC, 0.5% (w/v) SDS, and 100 μg/ml denatured, sonicated salmon sperm DNA) or low stringency (35% (v/v) formamide, 2× SSC, 0.5% (w/v) SDS, and 100 μg/ml denatured, sonicated salmon sperm DNA) for 74 h at 42 °C. Hybridization was performed using the 32P-labeled human HIP full-length cDNA as a probe (2–4 × 106 cpm/ml) in fresh solutions of identical composition for 16 h at 42 °C. The blots then were washed with 2× SSC, 0.1% (w/v) SDS for 5 min at 25 °C, then transferred to a fresh solution of identical composition at 42 °C for 2 h. The blots then were exposed to Kodak XAR films at −70 °C overnight. Positive plaques were isolated and further analyzed using the same hybridization and screening conditions. Nucleotide and protein sequence analyses were carried out using GCG and DNAStar programs and the data bases from GenBank (release 105.0), EMBL (release 51.0), and SWISS-PROT (release 35.0). A cDNA fragment corresponding to the entire coding sequence of the murine HIP/L29 was used to probe a Northern blot of purified poly(A)+ RNA (mouse MTN,CLONTECH) from various mouse tissues. After prehybridization at 68 °C for 30 min in ExpressHyb solution (CLONTECH), the α-32P-labeled murine HIP/L29 probe was added to fresh ExpressHyb solution (106cpm/ml) and allowed to interact with the membrane for 1 h. A 30-min rinse in 2× SSC (3 m NaCl, 0.3 m sodium citrate) and 0.1% (w/v) SDS at room temperature was followed by four washes of 20 min each in 0.1× SSC, 0.1% w/v SDS at 50 °C. The membrane was exposed to x-ray film at −70 °C with two intensifying screens for 6 h. The probe was removed by incubating the blot three times in sterile water containing 0.5% (w/v) SDS at 95–100 °C for 10 min. The blot was reprobed using a human cDNA probe for β-actin as a RNA quantification reference. This human probe is known to strongly cross-hybridize with mouse β-actin. Synthetic peptides of the following murine HIP/L29 sequences were constructed on a Vega 250 peptide synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) methodology (12Chang C.D. Meienhofer J. Int. J. Pept. Protein Res. 1978; 11: 246-249Crossref PubMed Scopus (312) Google Scholar): CMRFAKKHNKKGLKKM and CQPKPKVQTKAGAKA, corresponding to amino acids 43–57 and 120–133 of murine HIP/L29, respectively. These synthetic peptides were conjugated to keyhole limpet hemocyanin, using the Imject maleimide activated carrier protein kit (Pierce), and separately used for rabbit immunization following standard protocols (University of Texas M. D. Anderson Cancer Center Science Park, Bastrop, TX). Polyclonal antibodies were affinity-purified using synthetic peptides linked to maleimide-activated BSA (Pierce) and conjugated to cyanogen bromide-activated Sepharose (Sigma) using the manufacturer's protocol. Minced tissues were solubilized in sample extraction buffer: 4 m urea, 1% (w/v) SDS, 50 mm Tris (pH 7.0), 1% (v/v) β-mercaptoethanol, and 0.01% (v/v) phenylmethylsulfonyl fluoride. Solubilized samples were concentrated by precipitation with 10% (w/v) trichloracetic acid at 4 °C. Trichloracetic acid precipitates were centrifuged at 1200 ×g for 10 min at 4 °C, washed sequentially with 10% (w/v) trichloracetic acid and 100% acetone, and air dried. The pellets were dissolved in equal volumes of sample extraction buffer and sample buffer (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212382) Google Scholar), heated for 2 min at 90 °C, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples were resolved by SDS-PAGE on a 15% (w/v) acrylamide resolving gel as described (13Porzio M.A. Pearson A.M. Biochim. Biophys. Acta. 1977; 490: 27-34Crossref PubMed Scopus (520) Google Scholar, 14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212382) Google Scholar). After a brief rinse in transfer buffer (100 mm Tris base and 100 mm glycine, pH 9.2), the gel was transferred to a nitrocellulose membrane at 4 °C for 5 h at 40 V in a Transblot apparatus (Bio-Rad). The transferred blot was blocked with 1% (w/v) BSA in PAT buffer (PBS, 0.01% (w/v) sodium azide, and 0.05% (w/v) Tween 20) overnight at room temperature. The blot then was incubated with primary antibody diluted in 0.1% (w/v) BSA in PAT buffer for at least 4–6 h at room temperature, rinsed with PAT buffer three times for 5 min each, and incubated for at least 4–6 h with 6 μCi of 125I-protein A (45 mCi/mg) in 70 ml of 0.1% (w/v) BSA in PAT buffer. For staining of murine testis, uterus, and liver, these tissues were rapidly frozen in O.C.T. (Miles, Elkhart, IN) and 7-μm sections prepared at −20 °C on a Reichert Jung cryostat. These sections were fixed in 100% methanol for 10 min at room temperature, rehydrated in PBS for 5 min at room temperature, and immediately used for immunostaining. In all cases, the affinity purified murine HIP/L29 primary antibody generated against the peptide sequence, CQPKPKVQTKAGAKA, was used at a concentration of 0.05 mg/ml and the secondary antibody, fluorescein-conjugated donkey anti-rabbit Ig (Amersham Pharmacia Biotech), at a 1:10 dilution. NMuMG cells were grown to 70% confluence on a 100-mm tissue culture plate. Membranes were isolated by differential centrifugation. Briefly, cells were washed three times with PBS and released from the plate by incubation with 10 mm EDTA in PBS at 37 °C for 15 to 30 min. Cells were pelleted at 1000 × g for 10 min at 4 °C and resuspended in homogenizing buffer (0.25 m sucrose, 5 mm Tris-HCl (pH 7.4), 1 mm EDTA, 0.25 mm dithiothreitol) including protease inhibitors (15Farach M.C. Tang J.-P. Decker G.L. Carson D.D. Dev. Biol. 1987; 123: 401-410Crossref PubMed Scopus (100) Google Scholar) and homogenized on ice. The homogenate was centrifuged at 1000 ×g for 10 min at 4 °C. The 1000 × gsupernatant was centrifuged at 10,000 × g for 20 min at 4 °C. The 10,000 × g supernatant was centrifuged at 100,000 × g for 1 h at 4 °C. Samples of pellets and supernatants were analyzed by SDS-PAGE and Western blotting as described above. High speed (100,000 ×g) membrane fractions were divided into equal parts and extracted either with 0.15, 0.4, 0.8, 1.2, or 1.6 m NaCl in 0.25 m sucrose, 1 mm EDTA, 0.25 mmdithiothreitol, and 5 mm Tris (pH 7.4), incubated overnight at 4 °C, and centrifuged the next day at 100,000 ×g for 1 h. Supernatants were precipitated overnight at 4 °C by the addition of trichloracetic acid to a final concentration of 10% (w/v). Pellets were dissolved in 0.2 ml of sample extraction buffer and then precipitated and prepared for SDS-PAGE as described above. High speed (100,000 × g) particulate preparations were subjected to differential salt extraction with 0.4m NaCl followed by 0.8 m NaCl as described above. The proteins then were separated by two-dimensional gel electrophoresis (16O'Farrell P.Z. Goodman H.M. O'Farrell P.H. Cell. 1977; 12: 1133-1142Abstract Full Text PDF PubMed Scopus (2652) Google Scholar) and transferred to nitrocellulose as described above for Western blotting. The unblocked nitrocellulose was incubated with 125I-Bolton-Hunter reagent-derivatized Hp (17Raboudi N. Julian J. Rohde L.H. Carson D.D. J. Biol. Chem. 1992; 267: 11930-11939Abstract Full Text PDF PubMed Google Scholar) in 0.15m NaCl, in the presence of 500 μg/ml chondroitin sulfate C, overnight at 4 °C. The blot then was washed three times with PBS before drying for autoradiography. The same nitrocellulose membrane was blocked and then probed with affinity-purified antibodies directed against the peptide sequence, CMRFAKKHNKKGLKM, and binding subsequently visualized with a peroxidase ABC system using a diaminobenzidene substrate kit as described by the manufacturer's instructions (Vector Laboratories, Burlingame, CA). A parallel gel run under exactly the same conditions was silver-stained as described (18Wray W. Boulikas Q. Wray V.P. Hancock R. Anal. Biochem. 1981; 118: 197-203Crossref PubMed Scopus (2607) Google Scholar) to visualize the migration positions of all proteins on the gel. High speed (100,000 × g) membrane preparations were extracted overnight at 4 °C with 0.4 m NaCl in 5 mmTris (pH 8.0), and then centrifuged at 100,000 × g for 1.5 h. The 0.4 m NaCl-insoluble pellet was subsequently extracted with 0.8 m NaCl in 5 mmTris (pH 8.0) at 4 °C for 4 h and centrifuged 1.5 h at 100,000 × g. Murine HIP/L29 protein was eluted in the 0.8 m NaCl extract that was diluted subsequently to achieve a final concentration of 0.15 m NaCl, applied to a 0.5-ml pellet of pre-rinsed heparin-agarose (Sigma) and incubated overnight batchwise with constant rotary agitation at 4 °C. Stepwise salt elution from heparin-agarose was performed with NaCl extending from 0.15 to 2.0 m in 5 mm Tris pH 8.0. All fractions were trichloracetic acid-precipitated and prepared for SDS-PAGE and Western blotting as described above. PCR primers were made with BamHI adaptors to the murine HIP/L29 sequence nucleotides 21–504. The forward and reverse primers were: 5′-GGGGATCCCATGGCCAAGTCCAAG and 5′-GGGGATCCTTATGGGGCCTTCACAGGG, respectively. The amplified product containing the entire open reading frame of murine HIP/L29 was subcloned in-frame into theBamHI site of the Oligo-HIS vector, pV2a, as described previously (19Van Dyke M.W. Sirito M. Sawadago M. Gene (Amst.). 1992; 111: 99-104Crossref PubMed Scopus (132) Google Scholar). The product of this fusion added 17 N-terminal amino acids containing the oligohistidine motif. E. colitransformed with the plasmid construct were grown toA 600 = 0.62 prior to induction with 0.2 mm isopropyl-β-d-thiogalactopyranoside. The cells then were grown under induction for 5 h and harvested by centrifugation at 2000 × g for 15 min. The cells then were extracted with binding buffer (20 mm Tris-Cl, pH 8.0, 2 m NaCl, 0.1% (v/v) Triton X-100, and 20% (v/v) glycerol) under sonication. The ruptured cell lysate then was centrifuged at 10,000 × g for 10 min to remove any insoluble material. The cleared lysate then was poured onto a TALONTM (CLONTECH) cobalt metal affinity column that was pre-equilibrated with binding buffer. The proteins were allowed to bind batchwise for 10 min and washed three times for 10 min each, batchwise with binding buffer. The slurry then was loaded onto a column and washed with five column volumes of wash buffer (20 mm Tris-Cl, pH 8.0, 500 mm NaCl, 20% (v/v) glycerol, and 10 mm imidazole). The proteins finally were eluted with elution buffer (20 mm Tris-Cl, pH 8.0, 500 mm NaCl, 20% (v/v) glycerol, 100 mmimidazole) in 10 drop fractions into 50 μl of 200 mmEDTA, pH 7.0. After elution from the cobalt affinity column, column fractions were analyzed by SDS-PAGE and stained with Coomassie Blue to identify fractions containing murine HIP/L29. Murine HIP/L29-containing fractions were pooled and used in subsequent assays. [3H]Hp was used in solid-phase murine HIP/L29-binding assays performed in 96-well microassay plates. Approximately 600 ng of HIP, 600 ng of denatured BSA, or an equal volume of purified proteins from E. coli transformed with an expression vector containing murine HIP/L29 in the reverse direction were added to each well and dried at 37 °C overnight. Coating efficiency was determined by increasing the amount of HIP added to the well during the coating procedure until no further increase in [3H]Hp binding could be achieved. Under these conditions, essentially all of the recombinant human HIP up to 1.2 μg/well bound to the surface. The next day, each well was rinsed with PBS three times, and blocked with 100 μl of 0.1% (w/v) denatured BSA in a 37 °C incubator for at least 1 h. Each well then was rinsed with PBS three times, and then [3H]Hp (typically 1.0 × 105 dpm) was added in a final volume of 50 μl in PBS containing 0.1% (w/v) BSA, and incubated in a 37 °C incubator for 2 h. For glycosaminoglycan competition assays, unlabeled Hp (sodium salt, grade I-A from porcine intestinal mucosa), chondroitin sulfate C (from shark cartilage), dermatan sulfate (sodium salt, from porcine skin), heparan sulfate (sodium salt, from bovine kidney), keratan sulfate (sodium salt, from bovine cornea), and hyaluronic acid (from human umbilical chord) were added to the binding assays at a final concentration of 10 μg/ml. At the end of each experiment, unbound [3H]Hp was removed by rinsing three times with PBS. Bound [3H]Hp was extracted with 100 μl of extraction buffer (4 m guanidine HCl, 25 mmTris-Hcl, pH 8.0, 2.5 mm EDTA, and 0.02% (w/v) sodium azide) 3–4 h at 37 °C. All of the extract was counted in a Beckman scintillation counter. To identify a murine homologue to human HIP, a mouse uterine cDNA library was screened using a full-length human HIP cDNA (7Liu S. Smith S.E. Julian J. Rohde L.H. Karin N.J. Carson D.D. J. Biol. Chem. 1996; 271: 11817-11823Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Using both low and high stringency screening techniques, three different clones, 16–15, 18–14, and 18–4b, were identified (Fig. 1 A). These clones contained inserts with sizes of 970, 515, and 181 base pairs, respectively. Clones 18–14 and 18–4b had identical, overlapping sequences to the largest clone 16–15. Clone 16–15 contained the entire open reading frame of murine HIP/L29 (GenBank accession numberL08651) (11Rudert F. Garnier J. Schuhbaur B. Gene (Amst.). 1993; 133: 249-254Crossref PubMed Scopus (7) Google Scholar) that encodes a protein with a predicted molecular mass of 17,588 Da and an isoelectric point of 11.92 (Fig. 1 B). The only base pair substitution was a G → A substitution at position 504, reflecting a conserved change to the stop codon. The coding sequence for murine HIP/L29 cDNA was used to perform a search of the Swissprot protein data base. This search yielded five homologues to mouse HIP/L29 (Fig. 1 C). The sequences identified were putative HIP/L29 orthologues of rat (20Yuen-Ling C. Olvera J. Paz V. Wool I.G. Biochem. Biophys. Res. Commun. 1993; 192: 583-589Crossref PubMed Scopus (7) Google Scholar, 21Svoboda M. Ciccarelli E. Vandermeers-Piret M. Nagy A. Van De Weerdt C. Bollen A. Vandermeers A. Christophe J. Eur. J. Biochem. 1992; 203: 341-346Crossref PubMed Scopus (5) Google Scholar, 22Ostvold A.C. Hullstein I. Sletten K. FEBS Lett. 1992; 298: 219-222Crossref PubMed Scopus (9) Google Scholar) (Swissprot accession number p25886), human (7Liu S. Smith S.E. Julian J. Rohde L.H. Karin N.J. Carson D.D. J. Biol. Chem. 1996; 271: 11817-11823Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 23Law P.T.W. Tsui S.K.W. Lam W.Y. Luk S.C.W. Hwang D.M. Liew C.C. Lee C.Y. Fung K.P. Waye M.M.Y. Biochim. Biophys. Acta. 1996; 1305: 105-108Crossref PubMed Scopus (15) Google Scholar), Swissprot accession number p47914),Saccharomyces cerevisiae (24Murakami Y. Naitou M. Hagiwara H. Shibata T. Ozawa M. Sasanuma S. Sasanuma M. Tsuchiya Y. Soeda E. Yokoyama K. Nat. Genet. 1995; 10: 261-268Crossref PubMed Scopus (98) Google Scholar) (Swissprot accession number p05747), Saccharomyces pombe (25Otaka E. Higo K. Itoh T. Mol. Gen. Genet. 1983; 191: 519-524Crossref PubMed Scopus (35) Google Scholar) (Swissprot accession number q92366), and Drosophila (26Gaynor J.J. Fox M.G. Insect Biochem. Mol. Biol. 1997; 27: 233-238Crossref Scopus (0) Google Scholar, 27Fox M.G. Gaynor J.J. DNA Seq. 1997; 7: 1125-1235Google Scholar) (Swissprot accession number q24154), respectively, in the order of homology to murine HIP/L29. Several amino acids are conserved throughout the first 50 amino acids of the five sequences and additional amino acids are functionally conserved in this region. The Saccharomyces and Drosophila sequences all demonstrate large C-terminal truncations relative to mouse, rat, and human HIP/L29. Mouse shares 96.6% similarity to rat over 151 amino acids, 84.4% to human over 160 amino acids, 71.7% to S. cerevisiae over 53 amino acids, 63.2% to S. pombe over 57 amino acids, and 58.5% toDrosophila over 70 amino acids. The starting methionine of rat, human, S. cerevisiae, and the first three amino acids of S. pombe were absent from the proteins examined. The peptide sequences underlined (Fig. 1 B) were synthesized and antibodies generated toward these sequences. To determine the expression pattern of murine HIP in murine tissue, total, poly(A)+ RNA from heart, brain, spleen, lung, liver, skeletal muscle, and testis was probed with a 32P labeled murine HIP/L29 cDNA (Fig. 2). A single transcript with a size of approximately 1 kilobase was found in all tissues examined with variable transcript levels. Highest expression was observed in heart with the lowest expression observed in brain and spleen. The ubiquitously expressed β-actin probe generated a similar signal in each lane for the 2.0-kilobase α isoform of β-actin. In heart, skeletal muscle, and testis, the 1.8-kilobase γ isoform also was detected as described (28Giovanna P. Jardine K. McBurney M.W. Mol. Cell. Biol. 1991; 11: 4796-4803Crossref PubMed Scopus (73) Google Scholar) and is not related to mRNA degradation. The difference in signal intensities between the different lanes using β-actin as a control probe is due to the specialization of these tissues. These observations indicated that the large differences observed in HIP/L29 mRNA expression could not be accounted for by differences in gel loading or transfer efficiencies. Murine HIP/L29 protein expression also was examined by Western blot analysis. Testis, lung, serum, kidney, spleen, and liver protein extracts were examined (Fig. 3). Two different antibodies directed to predicted peptide motifs in murine HIP/L29 were generated and used for these studies as described in Fig. 1 B. Both antibodies recognized proteins with an apparent molecular mass of 24 kDa. Like its human counterpart, it is assumed that the molecular mass of murine HIP/L29 is higher than the calculated molecular mass of 17.6 kDa because of the large arginine and lysine content (26.9%) of the protein. A higher M r class of proteins were recognized by the anti-HIP peptide-2 antibody, but not the anti-HIP peptide-3 antibody, in liver and kidney (lanes 4and 6, Fig. 2 A). The identity of the largerM r proteins is not known. The 24-kDa protein displayed robust expression in testis, spleen, and liver with reduced expression in lung and was not detectable in whole mouse serum samples. Frozen sections of murine liver, uterus, and testis were probed with an antibody against HIP peptide-3 (Fig. 4). In liver, murine HIP/L29 is localized in discrete areas throughout the tissue (Fig. 4 A). Omisson of the primary antibody yielded no staining of similar frozen sections of liver (Fig. 4 B). In the uterus, murine HIP/L29 expression is largely confined to uterine and glandular epithelium (Fig. 4 D). Testis displayed strong, discrete staining of sperm tails and heads (Fig. 4, E and F). Collectively, these data demonstrate that murine HIP/L29 protein is expressed in a highly restricted pattern in several adult mouse tissues. To determine if murine HIP/L29, like human HIP, is associated with membranes (5Carson D.D. Rohde L.H. Surveyor G. Int. J. Biochem. 1994; 26: 1269-1277Crossref PubMed Scopus (27) Google Scholar), a subcellular fractionation procedure was used to examine HIP subcellular localization by Western blot analysis (Fig. 5). Like human HIP, murine HIP/L29 is enriched in the 100,000 × g particulate fraction, suggesting that a portion of the NMuMg's murine HIP/L29 population is membrane-associated. Murine HIP/L29, as well as human HIP, lacks a potential transmembrane spanning peptide motif. Therefore, it was considered that murine HIP/L29 was peripherally associated with membranes. To test this hypothesis, the 100,000 × g particulate fraction was extracted with NaCl concentrations up to 1.6 m. Murine HIP/L29 was quantitatively eluted from membranes with NaCl concentrations of 0.8 m or above (Fig. 6). These characteristics were consistent with the properties expected of a peripheral membrane protein. To test the function of murine HIP/L29 as a Hp-binding protein, three procedures were employed. In the first, HIP/L29-containing fractions solubilized by 0.8 mNaCl extraction were separated by two-dimensional PAGE. Many proteins were detected in this fraction by silver staining (Fig. 7 A); however, only a subset of these proteins directly bound 125I-Hp in an overlay assay (Fig. 7 B). Out of these 125I-Hp-binding proteins, only one was recognized by anti-HIP/L29 (Fig. 7 C). This protein migrates at a highly basic pH with an apparent molecular mass of 24 kDa. Parallel 125I-Hp overlays were done in the presence of either 500 μg/ml unlabeled chondroitin sulfate C or 500 μg/ml unlabeled Hp. Although 125I-Hp binding was markedly diminished in the presence of 500 μg/ml unlabeled Hp, no reduction was observed in the presence of 500 μg/ml chondroitin sulfate (data not shown). These data indicated that direct binding of125I-Hp to murine HIP/L29 was highly selective for Hpversus other polyanions. The second procedure involved fractionation of the 0.8 mNaCl solubilized HIP/L29 on Hp-agarose (Fig. 8). The relative affinity of murine HIP was examined by determining the salt concentration required for elution. In this procedure, murine HIP bound Hp-agarose efficiently at physiological salt concentrations and eluted sharply and completely with 0.75 m NaCl. These observations demonstrated that murine HIP/L29 bound Hp-agarose avidly at physiological pH. A third procedur
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