Absence of Post-translational Aspartyl β-Hydroxylation of Epidermal Growth Factor Domains in Mice Leads to Developmental Defects and an Increased Incidence of Intestinal Neoplasia
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m110389200
ISSN1083-351X
AutoresJoseph Dinchuk, Richard J. Focht, Jennifer Kelley, Nancy Henderson, Nina Zolotarjova, Richard Wynn, Nicola T. Neff, John Link, Reid Huber, Timothy C. Burn, Mark Rupar, Mark R. Cunningham, Bernard Selling, Jianhong Ma, Andrew Stern, Gregory Hollis, Robert B. Stein, Paul A. Friedman,
Tópico(s)Developmental Biology and Gene Regulation
ResumoThe BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear. To evaluate the role of BAH in vivo, the catalytic domain of BAH was specifically targeted such that the coding regions of junctin and humbug remained undisturbed. BAH null mice lack measurable BAH protein in several tissues, lack aspartyl β-hydroxylase activity in liver preparations, and exhibit no hydroxylation of the epidermal growth factor (EGF) domain of clotting Factor X. In addition to reduced fertility in females, BAH null mice display several developmental defects including syndactyly, facial dysmorphology, and a mild defect in hard palate formation. The developmental defects present in BAH null mice are similar to defects observed in knock-outs and hypomorphs of the Notch ligand Serrate-2. In this work, β-hydroxylation of Asp residues in EGF domains is demonstrated for a soluble form of a Notch ligand, human Jagged-1. These results along with recent reports that another post-translational modification of EGF domains in Notch gene family members (glycosylation by Fringe) alters Notch pathway signaling, lends credence to the suggestion that aspartyl β-hydroxylation may represent another post-translational modification of EGF domains that can modulate Notch pathway signaling. Previous work has demonstrated increased levels of BAH in certain tumor tissues and a role for BAH in tumorigenesis has been proposed. The role of hydroxylase in tumor formation was tested directly by crossing BAH KO mice with an intestinal tumor model,APCmin mice. Surprisingly, BAH null/APCmin mice show a statistically significant increase in both intestinal polyp size and number when compared with BAH wild-type/APCmincontrols. These results suggest that, in contrast to expectations, loss of BAH catalytic activity may promote tumor formation. The BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear. To evaluate the role of BAH in vivo, the catalytic domain of BAH was specifically targeted such that the coding regions of junctin and humbug remained undisturbed. BAH null mice lack measurable BAH protein in several tissues, lack aspartyl β-hydroxylase activity in liver preparations, and exhibit no hydroxylation of the epidermal growth factor (EGF) domain of clotting Factor X. In addition to reduced fertility in females, BAH null mice display several developmental defects including syndactyly, facial dysmorphology, and a mild defect in hard palate formation. The developmental defects present in BAH null mice are similar to defects observed in knock-outs and hypomorphs of the Notch ligand Serrate-2. In this work, β-hydroxylation of Asp residues in EGF domains is demonstrated for a soluble form of a Notch ligand, human Jagged-1. These results along with recent reports that another post-translational modification of EGF domains in Notch gene family members (glycosylation by Fringe) alters Notch pathway signaling, lends credence to the suggestion that aspartyl β-hydroxylation may represent another post-translational modification of EGF domains that can modulate Notch pathway signaling. Previous work has demonstrated increased levels of BAH in certain tumor tissues and a role for BAH in tumorigenesis has been proposed. The role of hydroxylase in tumor formation was tested directly by crossing BAH KO mice with an intestinal tumor model,APCmin mice. Surprisingly, BAH null/APCmin mice show a statistically significant increase in both intestinal polyp size and number when compared with BAH wild-type/APCmincontrols. These results suggest that, in contrast to expectations, loss of BAH catalytic activity may promote tumor formation. aspartyl β-hydroxylase epidermal growth factor 1,4-piperazinediethanesulfonic acid Aspartyl β-hydroxylase (BAH)1 catalyzes the post-translational hydroxylation of aspartic acid or asparagine residues contained within epidermal growth factor (EGF) domains of proteins. The consensus sequence for aspartyl/asparaginyl hydroxylation has been found in a wide range of proteins including clotting factors, Notch receptors, and their ligands, ligands of the tyro-3/Axl family of receptor tyrosine kinases and structural proteins of the extracellular matrix (1.Stenflo J. Blood. 1991; 78: 1637-1651Crossref PubMed Google Scholar, 2.Rebay I. Fleming R.J. Fehon R.G. Cherbas L. Artavanis-Tsakonas S. Cell. 1991; 67: 689-699Abstract Full Text PDF Scopus (597) Google Scholar, 3.Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Hanford P.A. Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 4.Goruppi S. Yamane H. Marcandalli P. Garcia A. Clogston C. Gostissa M. Varnum B. Schneider C. FEBS Lett. 1997; 415: 59-63Crossref PubMed Scopus (19) Google Scholar, 5.Nakamura T. Ruiz-Lozano P. Lindner V. Yabe D. Taniwaki M. Furukawa Y. Kobuke K. Tashiro K. Lu Z. Andon N. Schaub R. Matsumori A. Sasayama S. Chien K.R. Honjo T. J. Biol. Chem. 1999; 274: 22476-22483Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). In addition, the gene encoding BAH is conserved from Drosophila to man (6.Dinchuk J.E. Henderson N.L. Burn T.C. Huber R. Ho S.P. Link J. O'Neil K.T. Focht R.J. Scully M.S. Hollis J.M. Hollis G.F. Friedman P.A. J. Biol. Chem. 2000; 275: 39543-39554Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). To date, the biological role for this post-translational modification has remained elusive. Recent studies on the genomic organization of the BAH locus revealed that BAH contains 24 exons and spans over 200 kilobases of genomic DNA (6.Dinchuk J.E. Henderson N.L. Burn T.C. Huber R. Ho S.P. Link J. O'Neil K.T. Focht R.J. Scully M.S. Hollis J.M. Hollis G.F. Friedman P.A. J. Biol. Chem. 2000; 275: 39543-39554Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). These studies yielded the surprising observation that two additional proteins are produced from this locus. One of these proteins, humbug (junctate), shares an identical NH2-terminal half of the protein with BAH, but lacks the entire 52-kDa COOH-terminal catalytic domain (6.Dinchuk J.E. Henderson N.L. Burn T.C. Huber R. Ho S.P. Link J. O'Neil K.T. Focht R.J. Scully M.S. Hollis J.M. Hollis G.F. Friedman P.A. J. Biol. Chem. 2000; 275: 39543-39554Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 7.Treves S. Feriotto G. Moccagatta L. Gambari R. Zorzato F. J. Biol. Chem. 2000; 275: 39555-39568Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Humbugencodes a type II membrane protein with a short putative amino-terminal cytoplasmic domain, a transmembrane domain, and a highly charged lumenal domain. The third protein encoded by this locus, junctin, has been previously described and is known to be an integral part of the ryanodine receptor complex (8.Jones L.R. Zhang L. Sanborn K. Jorgensen A.O. Kelley J. J. Biol. Chem. 1995; 270: 30787-30796Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 9.Zhang L. Schmeisser G. Kobayashi Y.M. Jones L.R. J. Biol. Chem. 1997; 272: 23389-23397Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). Junctin shares a common NH2-terminal end with BAH and humbug that corresponds to the cytoplasmic domain, the transmembrane domain and the first 42 amino acids on the endoplasmic reticulum lumenal side of the membrane (6.Dinchuk J.E. Henderson N.L. Burn T.C. Huber R. Ho S.P. Link J. O'Neil K.T. Focht R.J. Scully M.S. Hollis J.M. Hollis G.F. Friedman P.A. J. Biol. Chem. 2000; 275: 39543-39554Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Alternative splicing leads to the addition of sequence from an alternative COOH-terminal exon that is specific to junctin (6.Dinchuk J.E. Henderson N.L. Burn T.C. Huber R. Ho S.P. Link J. O'Neil K.T. Focht R.J. Scully M.S. Hollis J.M. Hollis G.F. Friedman P.A. J. Biol. Chem. 2000; 275: 39543-39554Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Alternative splicing of different exons of the BAH locus also gives rise to minor transcriptional forms of these three proteins. 2J. E. Dinchuk, R. J. Focht, J. A. Kelley, N. L. Henderson, R. M. Huber, T. C. Burn, G. F. Hollis, R. B. Stein, and P. A. Friedman, unpublished observations.2J. E. Dinchuk, R. J. Focht, J. A. Kelley, N. L. Henderson, R. M. Huber, T. C. Burn, G. F. Hollis, R. B. Stein, and P. A. Friedman, unpublished observations. While a great deal is known about the biochemical activity of BAH (10.Gronke R.S. Welsch D.J. VanDusen W.J. Garsky V.M. Sardana M.K. Stern A.M. Friedman P.A. J. Biol. Chem. 1990; 265: 8558-8565Abstract Full Text PDF PubMed Google Scholar, 11.Wang Q. Van Dusen W.J. Petroski C.J. Garsky V.M. Stern A.M. Friedman P.A. J. Biol. Chem. 1991; 266: 14004-14010Abstract Full Text PDF PubMed Google Scholar, 12.Jia S. VanDusen W.J. Diehl R.E. Kohl N.E. Dixon R.A. Elliston K.O. Stern A.M. Friedman P.A. J. Biol. Chem. 1992; 267: 14322-14327Abstract Full Text PDF PubMed Google Scholar, 13.Monkovic D.D. VanDusen W.J. Petroski C.J. Garsky W.M. Sardana M.K. Zavodszky P. Stern A.M. Friedman P.A. Biochem. Biophys. Res. Commun. 1992; 189: 233-241Crossref PubMed Scopus (28) Google Scholar, 14.Jia S. McGinnis K. VanDusen W.J. Burke C.J. Kuo A. Griffin P.R. Sardana M.K. Elliston K.O. Stern A.M. Friedman P.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7227-7231Crossref PubMed Scopus (50) Google Scholar, 15.McGinnis K. Ku G.M. VanDusen W.J. Fu J. Garsky V. Stern A.M. Friedman P.A. Biochem. 1996; 35: 3957-3962Crossref PubMed Scopus (39) Google Scholar, 16.McGinnis K. Ku G.M. Fu J. Stern A.M. Friedman P.A. Biochim. Biophys. Acta. 1998; 1387: 454-456Crossref PubMed Scopus (4) Google Scholar), little is known about the biological role of the hydroxylation of EGF domains. Initial work focused on coagulation cascade proteases that are known to contain varying levels of hydroxylation of their EGF domains (17.Derian C.K. VanDusen W. Przysiecki C.T. Walsh P.N. Berkner K.L. Kaufman R.J. Friedman P.A. J. Biol. Chem. 1989; 264: 6615-6618Abstract Full Text PDF PubMed Google Scholar, 18.Gronke R.S. VanDusen W.J. Garsky V.M. Jacobs J.W. Sardana M.K. Stern A.M. Friedman P.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 3609-3613Crossref PubMed Scopus (55) Google Scholar, 19.Nelson R.M. VanDusen W.J. Friedman P.A. Long G.L. J. Biol. Chem. 1991; 266: 20586-20589Abstract Full Text PDF PubMed Google Scholar, 20.Sunnerhagen M.S. Persson E. Dahlqvist I. Drakenberg T. Stenflo J. Mayhew M. Robin M. Handford P. Tilley J.W. Campbell I.D. Brownlee G.G. J. Biol. Chem. 1993; 268: 23339-23344Abstract Full Text PDF PubMed Google Scholar). These studies were unable to demonstrate altered activity of the proteases when the level of hydroxylation was varied. Multiple additional roles for hydroxylase have been postulated. One proposed role is that hydroxylase may modify signaling through the Notch pathway by direct hydroxylation of Notch receptors and/or their ligands, all of which contain multiple EGF domains with consensus hydroxylation sequences (13.Monkovic D.D. VanDusen W.J. Petroski C.J. Garsky W.M. Sardana M.K. Zavodszky P. Stern A.M. Friedman P.A. Biochem. Biophys. Res. Commun. 1992; 189: 233-241Crossref PubMed Scopus (28) Google Scholar, 21.Lavoissiere L. Jia S. Nishiyama M. de la Monte S.M. Stern A.M. Wands J.R. Friedman P.A. J. Clin. Invest. 1996; 98: 1313-1323Crossref PubMed Scopus (121) Google Scholar). There has been no direct proof that BAH plays a role in this pathway or even that these proteins are hydroxylated. Another proposed role for aspartyl β-hydroxylase, and humbug, was derived directly from the observation that these two proteins are encoded by the same locus as junctin and share significant coding regions (6.Dinchuk J.E. Henderson N.L. Burn T.C. Huber R. Ho S.P. Link J. O'Neil K.T. Focht R.J. Scully M.S. Hollis J.M. Hollis G.F. Friedman P.A. J. Biol. Chem. 2000; 275: 39543-39554Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 7.Treves S. Feriotto G. Moccagatta L. Gambari R. Zorzato F. J. Biol. Chem. 2000; 275: 39555-39568Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Direct proof of interaction of BAH and humbug with the ryanodine receptor has not been shown, but preliminary experiments suggest that overexpression of humbug in in vitro systems can alter Ca2+ movement in cell culture systems (7.Treves S. Feriotto G. Moccagatta L. Gambari R. Zorzato F. J. Biol. Chem. 2000; 275: 39555-39568Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). BAH overexpression has also been associated with epithelial malignancies. Initial observations suggested that BAH is overexpressed in cholangiocarcinoma and liver cancer (21.Lavoissiere L. Jia S. Nishiyama M. de la Monte S.M. Stern A.M. Wands J.R. Friedman P.A. J. Clin. Invest. 1996; 98: 1313-1323Crossref PubMed Scopus (121) Google Scholar). These studies measured BAH levels by utilizing both polyclonal antibodies directed against a fusion protein coding for humbug and the monoclonal antibody FB-50, which has now been demonstrated to detect both BAH and humbug (6.Dinchuk J.E. Henderson N.L. Burn T.C. Huber R. Ho S.P. Link J. O'Neil K.T. Focht R.J. Scully M.S. Hollis J.M. Hollis G.F. Friedman P.A. J. Biol. Chem. 2000; 275: 39543-39554Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Because these antibody reagents recognize both BAH and humbug, it is difficult to tell if one or both of these proteins are up-regulated in these malignancies. Recent studies using NIH 3T3 cells transfected with murine and human BAH suggested a more direct role for BAH in transformation (22.Ince N. de la Monte S.M. Wands J.R. Cancer Res. 2000; 60: 1261-1266PubMed Google Scholar). Because it has been difficult to establish the biological role of BAH by direct biochemical approaches, a genetic approach was taken to assess the role of BAH in vivo. Transgenic mice were generated in which the catalytic activity of BAH was removed by deletion of exons 22 and 23 using homologous recombination. These exons were targeted for removal because they are required for BAH catalytic activity, but are spatially well separated from the coding exons of humbug and junctin. BAH catalytic null animals were evaluated for developmental and reproductive defects. While BAH null animals are viable, they exhibit multiple developmental abnormalities reminiscent of those seen in animals carrying Notch ligand mutations and have altered reproductive capacity. To provide additional insight into a possible role for BAH in tumorigenesis, these mice were crossed with mice carrying the APCmin mutation that leads to multiple intestinal polyp formation. BAH null mice were compared with BAH wild-type animals carrying the APCmin mutation. Analysis of BAH catalytic null animals carrying the APCmin mutation revealed a significant increase in region-specific polyp size and frequency when compared with BAH wild-type animals. A region of mouse BAH known to be critical for catalytic activity (14.Jia S. McGinnis K. VanDusen W.J. Burke C.J. Kuo A. Griffin P.R. Sardana M.K. Elliston K.O. Stern A.M. Friedman P.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7227-7231Crossref PubMed Scopus (50) Google Scholar, 15.McGinnis K. Ku G.M. VanDusen W.J. Fu J. Garsky V. Stern A.M. Friedman P.A. Biochem. 1996; 35: 3957-3962Crossref PubMed Scopus (39) Google Scholar) was targeted for removal by homologous recombination in AB2.2 ES cells. Southern blotting and long range PCR of DNA derived from ES cells were used to confirm correct targeting at this locus (Fig. 1, a and b). Targeted ES cells were injected into donor C57/BL6 blastocysts to produce founder chimeric mice. Founder chimeric mice were mated to produce germ-line transmission of the knock-out. All mice were housed in a fully accredited (by the American Association for the Accreditation of Animal Care International), specific pathogen-free facility at the Bristol-Myers Squibb Pharma Research Labs, Inc. (Wilmington, DE). C57BL/6J-APCmin mice were purchased from The Jackson Laboratory (Bar Harbor, ME), stock number 002020, and maintained by crossing APCmin/Apc+ males with C57BL/6NTac females purchased from Taconic Farms (Germantown, NY), model number B6. BAH(+/−) APCmin/Apc+ mice were generated by mating APCmin/Apc+ males with BAH (+/−) or (−/−) females having a mixed strain background (129S6/SvEv × C57BL/6NTac). Male BAH(+/−)APCmin/Apc+ mice were mated with BAH(+/−) females to produce BAH(−/−) APCmin/Apc+ and BAH(+/+)APCmin/Apc+ mice for study. Intestines were harvested from 110-day-old BAH(−/−)APCmin/Apc+, and BAH(+/+) APCmin/Apc+ male and female mice after CO2 euthanasia. For approximately one-third of the BAH(−/−) APCmin/Apc+ mice it was necessary to harvest the tissue between days 84 and 108 so as to avoid premature death because of the severity of the disease developing in these animals. Entire intestinal tracts were removed and flushed with phosphate-buffered saline from stomach to cecum, and from anus to cecum. The gut epithelium was distended and fixed by filling the tract with phosphate-buffered saline-buffered 10% formalin (pH 7.0). The small intestines were divided into three sections as outlined below and polyps were scored along their entire lengths. Proximally, the duodenum included the region 4 cm immediately distal to the stomach. Distally, the ileum encompassed the section 4 cm immediately proximal to the cecum. The middle section (jejunum) connected the duodenum and ileum and varied in length from 10.5 to 30 cm with an average length of 22.1 cm in BAH(+/+) mice and 20.7 cm in BAH(−/−) mice. Polyps were also counted along the entire length of the colon. Each section was opened longitudinally and polyps were counted and measured under a stereomicroscope at ×10–60 magnification. Tumors from 21 null and 22 wild-type mice were counted and measured by examination at ×30 magnification from four regions of the gastrointestinal tract: duodenum, jejunum, ileum, and the colon. Count data and tumor size data were analyzed using a square root transformation to stabilize the variance. The three primer pairs used for genotyping APCmin mice were: IMR033 5′-GCCATCCCTTCCGTTAG-3′, IMR034 5′-TTCCATTTGGCATAGGC-3′, and IMR758 5′-TTCTGAGAAAGACAGAATTA-3′. The PCR conditions used are those recommended by the Induced Mutant Resource of The Jackson Laboratory. To screen for the BAH catalytic exons 22–23 knockout, a forward primer at the 5′ end outside of the resistance cassette (Spe-2, 5′-TCTGTGTACTACAATAATTTGGC-3′) and a reverse primer inside the resistance cassette (neo805P, 5′-AGGACATAGCGTTGGCTA-3′) were used (Fig. 1a). For the wild-type region the same forward primer, Spe-2, was used in combination with a primer within exon 22 (22-2, 5′-AGTAAAGCACAGGTCTTAGGC-3′). The wild-type product is ∼800 bp long and the disrupted product is ∼400 bp long (Fig. 1a). By evaluating both of these PCR reaction products, the KO status of the mice can be determined. Taq DNA polymerase with associated buffer and MgCl2 from Invitrogen (Gaithersburg, MD) were used for conventional PCR according to manufacturer's instructions for both the APCmin and BAH wild-type control PCR. For amplification of the BAH catalytic KO region, Taq DNA polymerase was used with Optitaq G buffer (Qbiogene, Carlsbad, CA). Real-time PCR was performed essentially as described (23.Gibson U.E. Heid C.A. Williams P.M. Genome Res. 1996; 6: 995-1001Crossref PubMed Scopus (1771) Google Scholar). Primers and probes were synthesized and purified by Biosearch Technologies, Inc. (Novato, CA). All probes, with the exception of the 18 S probe, were modified at the 5′ end with the reporter dye 6-carboxyfluorescein aminohexylamidite, and at the 3′ end with the quencher dye 6-carboxytetramethyl rhodamine (Biosearch Technologies Inc.). The 5′ end of the 18 S probe was modified with VIC (Applied Biosystems, Foster City, CA). For Asph exon 5a (junctin), primers ACCCATCAAAGAAGAGCTGAAGA, CCCCTTCCCTCTATCCTCCTG, and probe CCCTGCCTTCGCTCTTCATTCTTGCT were used. For AspH exon 14a (humbug), primers GGAATTCAGGGTGTATGAGAAACAG, CCAGTGTATAAAGGAAGAGGCTCATC, and probe CCCAGAGTTTGCTGCTGGGTCCAA were used. For AspH exon 23 (catalytic domain), primers TGCACCCTGGAACTCATGTG, GGATCACTAACCCCAGATGCAT, and probe CAGGACCCACAAACTGCAGGCTCC were used. For 18 S rRNA, primers CGGCTACCACATCCAAGGAA, GCTGGAATTACCGCGGCT, and probe TGCTGGCACCAGACTTGCCCTC were used. Total RNA was prepared from murine heart tissue using the RNeasy purification system according to the manufacturer's (Qiagen, Valencia, CA) instructions. cDNA synthesis was performed using the Advantage RT-PCR kit (CLONTECH, Palo Alto, CA) according to the manufacturer's instructions. Briefly, 1 μg of total RNA was DNase I-treated and reverse transcribed using random hexamers and Moloney murine leukemia virus reverse transcriptase. For Taqman-based real-time PCR expression profiling, 25 ng of each cDNA was added to the Taqman Universal PCR Master Mix (PerkinElmer Life Science, Foster City, CA) along with 900 nm of each primer and 200 nmof probe according to the manufacturer's instructions. Real-time fluorescence monitoring was performed with the PerkinElmer 7700. Standard curves were generated for each transcript using a serial dilution of cDNA. Relative abundance was then determined by comparing the cycle threshold values for each reaction with this standard curve. Abundance levels calculated from negative control reactions performed in the absence of reverse transcriptase were then subtracted from experimental sample abundance. Input levels of cDNA were corrected for by normalizing all data to 18 S ribosomal RNA levels. All expression measurements were performed in quadruplicate using two independently generated cDNA samples. Mouse liver specimens (∼30 mg each) were homogenized on ice with 200 μl of buffer A1 (buffer A: 50 mm Tris-HCl, pH 7.5, 50 mm NaCl, 0.9% Nonidet P-40, 10 mmdithiothreitol, 1 mm EDTA, 1 mg/ml Pefabloc SC, 1 mg/ml aprotinin and 10 mg/ml leupeptin) in a 2-ml tube using a Tissuemizer (Becton Dickinson) running at ∼11,000 rpm for 25 s. All subsequent procedures were performed at 4 °C. Cell lysates were diluted by adding an additional 200 μl of buffer A and were then loaded onto QIAshredder spin columns (Qiagen Inc., Valencia, CA) to shear high molecular weight DNA. After centrifuging in microcentrifuge tubes at 14,000 rpm for 15 min, supernatants were transferred to 1.5-ml Eppendorf tubes. The eluates were adjusted to a volume of ∼0.5 ml with buffer A and were re-centrifuged at 14,000 rpm for another 30 min. The protein concentrations of the supernatants were determined by the Lowry method. Three mg of total protein (∼150–300 μl) from each supernatant was added to 0.5 ml of 50% SP-Sepharose High Performance resin pre-equilibrated with buffer B (buffer B: 50 mmTris-HCl, pH 7.5, 50 mm NaCl, 0.9% Nonidet P-40, 10 mm dithiothreitol, 0.1 mg/ml Pefabloc SC, 1 mg/ml aprotinin, and 1 mg/ml leupeptin). The total volume including resin was adjusted to 1 ml with buffer B. These mixtures were equilibrated on a roller for 40 min and were then centrifuged briefly at ∼1000 rpm for 10–15 s. After 4 successive 1-ml washes in buffer B, bound proteins were eluted with 4 successive washes of 0.25 ml of buffer C (buffer C: 50 mm Tris-HCl, pH 7.5, 1 M NaCl, 5% glycerol, 10 mm dithiothreitol, 1 mg/ml aprotinin, 0.1 mg/ml Pefabloc SC, and 1 mg/ml leupeptin) each. All buffers were stored at 4 °C. Each elution was performed by mixing the resin with buffer C on a roller for 5 min. Eluted proteins were pooled to reach a final volume of 1 ml/tissue sample and were stored at −70 °C awaiting determination of enzymatic activity. To determine the recovery of aspartyl β-hydroxylase activity during the purification process, a known amount of human 52-kDa aspartyl β-hydroxylase catalytic domain (14.Jia S. McGinnis K. VanDusen W.J. Burke C.J. Kuo A. Griffin P.R. Sardana M.K. Elliston K.O. Stern A.M. Friedman P.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7227-7231Crossref PubMed Scopus (50) Google Scholar)2 was added to a control preparation prior to homogenization. Based upon the recovery of activity in this sample the recovery of aspartyl β-hydroxylase activity after purification from mouse tissue samples was determined to be 70–80%. Aspartyl β-hydroxylase activity was assayed as described previously (10.Gronke R.S. Welsch D.J. VanDusen W.J. Garsky V.M. Sardana M.K. Stern A.M. Friedman P.A. J. Biol. Chem. 1990; 265: 8558-8565Abstract Full Text PDF PubMed Google Scholar, 11.Wang Q. Van Dusen W.J. Petroski C.J. Garsky V.M. Stern A.M. Friedman P.A. J. Biol. Chem. 1991; 266: 14004-14010Abstract Full Text PDF PubMed Google Scholar, 12.Jia S. VanDusen W.J. Diehl R.E. Kohl N.E. Dixon R.A. Elliston K.O. Stern A.M. Friedman P.A. J. Biol. Chem. 1992; 267: 14322-14327Abstract Full Text PDF PubMed Google Scholar). The first EGF-like domain of human factor IX with aspartic acid at position 18 was used as the substrate. Incubations were carried out in a final volume of 40 μl in 1.5-ml siliconized Eppendorf tubes at 37 °C for 30 min. Final concentrations of reagents were 50 mm PIPES, pH 7.0, 100 mmferrous ammonium sulfate, 20 mmα-[1-14C]ketoglutaric acid (α-[1-14C]ketoglutaric acid (54.5 mCi/mmol) (PerkinElmer Life Science), 120 mm EGF substrate, and 0.2 mg/ml bovine serum albumin. A linear response was obtained when between 12 and 24 μl of partial purified aspartyl β-hydroxylase was used. Mouse blood was collected by direct cardiac puncture into anticoagulant (1/10th of the final volume). The anticoagulant consisted of 84 mg of citric acid, 177 mg of trisodium citrate, 116 mg of benzamidine, 2 mg of soybean trypsin inhibitor, and 100 units of heparin in a final volume of 10 ml. Plasma was prepared by centrifugation at 3000 × g for 5 min. 1 mbarium chloride was added to the plasma slowly with stirring during 2 h of incubation at 4 °C. For 1 ml of plasma, 90 μl of barium chloride was added. This mixture was incubated overnight at 4 °C with mixing. After centrifugation for 15 min at 3000 ×g, pellets were washed 3 times with 300 μl of 0.9% NaCl, 5 mm benzamidine. Final pellets were dissolved in 120 μl of 0.2 m EDTA, 5 mm benzamidine, pH 7.4, and incubated for 1 h with mixing at 4 °C. The protein mixture was dialyzed overnight at 4 °C against 50 mm Tris-HCl, 150 mm NaCl, 5 mm benzamidine, pH 7.5. After dialysis, proteins were aliquoted and stored at −70 °C. The protein mixture was further resolved by high performance liquid chromatography using a 2.1 × 50-mm Vydac C4 column. The Vydac C4 column was equilibrated in 0.05% trifluoroacetic acid, 10% acetonitrile, and between 50 and 100 μl of protein mixture was loaded. Subsequently, a linear gradient of acetonitrile from 10 to 47.5% was applied at an increasing rate of ∼0.23% per minute. Protein fractions from 1 to 10 were collected and analyzed on an SDS-PAGE gel. Fraction 1 was eluted from the column at an acetonitrile concentration of 36%. Fraction 1 contained a single protein band on SDS-PAGE. This band was cut out, minced, and the protein was eluted and sequenced. The NH2-terminal sequence of this band corresponded to the reported NH2-terminal sequence of mouse Factor X (data not shown). All glass tubes and vials were pyrolyzed at 500 °C for at least 4 h. Protein samples were dried in the glass tubes in a Speedvac (Savant Instruments Inc., Hicksville, NY). 500 μl of 6n HCl (Pierce Biochemicals, Rockford, IL) was added to each hydrolysis vial. Acid hydrolysis tubes were incubated for 20 h at 110 °C under an argon atmosphere. After hydrolysis, samples were dried and processed according to Waters AccQ-Fluor Reagent kit protocol. Derivatized samples (100 μl) were dried in the Speedvac, dissolved in 20 μl of 10% acetonitrile, and loaded onto an high performance liquid chromatography column under conditions and gradient specified by Waters (Millipore Corporation, Milford, MA). For Hya (β-hydroxyaspartic acid) calculations, various amounts of e-Hya (24.Przysiecki C.T. Staggers J.E. Ramjit H.G. Musson D.G. Stern A.M. Bennett C.D. Friedman P.A. Proc. Nat. Acad. Sc. U. S. A. 1987; 84: 7856-7860Crossref PubMed Scopus (59) Google Scholar) were spiked into amino acid analysis standards and analyzed for the peak area and retention time under experimental conditions. Wild-type and knock-out mouse tissues were collected in separate vials and placed into a dry ice bath. Thawed tissues were washed briefly in buffer (50 mm Tris-HCl, pH 7.5, 2 mmEDTA, 150 mm NaCl, 0.5 mm dithiothreitol) plus 1 tablet of “Complete” protease inhibitors (Roche Molecular Biochemicals)/50 ml of buffer. Tissues were homogenized for 30 s on ice in extraction buffer (62.5 mm Tris-HCl, pH 7.5, 2% SDS, 10% glycerol, 2 tablets of Complete per 50 ml of extraction buffer). Extracts were incubated for 30 min at 4 °C on a rotating wheel, centrifuged for 15 min at 3000 × g, aliquoted, and stored at −70 °C. For Western blot analysis, 50 μg of wild-type and knock-out tissue proteins were separated by 4–20% SDS-PAGE (Novex, Invitrogen, Carlsbad, CA). Proteins were transferred onto polyvinylidene difluoride membranes for 2 h at 50 V with an ice pack cooling unit using 10 mm CAPS-NaOH, pH 11.0, 10% methanol as a transfer buffer. Membranes were blocked in 5% milk, phosphate-buffered saline, pH 7.0, for 2 h. Incubation with primary polyclonal antibody was carried out overnight at 4 °C. Antibodies were raised in rabbits against the catalytic domain of human BAH (14.Jia S. McGinnis K. VanDusen W.J
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