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PubMed2002

Expression, purification, and characterization of human dipeptidyl peptidase IV/CD26 in Sf9 insect cells.

The human dipeptidyl peptidase IV/CD26 (DPPIV/CD26) is a multifunctional type-II membrane bound glycoprotein. As a receptor of collagen I and fibronectin it mediates cell-cell and cell-matrix adhesion, and by interacting with extracellular adenosine deaminase and CD45 it is involved in regulatory and costimulatory events in the immune system. DPPIV/CD26 has a very distinct substrate specificity, and is potentially capable of truncating many cytokines, chemokines, and peptide hormones. In this study, we describe the overexpression, purification, and characterization of human DPPIV/CD26 in Spodoptera frugiperda (Sf9) cells, using the baculovirus system. Overexpression of DPPIV/CD26 was confirmed by measurement of its peptidase specificity, SDS-PAGE, and Western blot analyses. Expression rates were between 6.4 and 17.6 mg protein per liter suspension culture (1.5 x 10(9)cells). The N-linked oligosaccharide composition was examined and compared with that of mammalian cell-expressed DPPIV/CD26. Two-step purification by immunoaffinity chromatography and size-exclusion fast protein liquid chromatography (SE-FPLC) led to highly stable protein with significant peptidase activity. A subsequent gel filtration step on a Superdex 200 column yielded 2mg homogeneous dimeric DPPIV/CD26 (per liter insect cell culture) for crystallographic studies. Protein homogeneity was confirmed by silver staining of non-denaturating PAGE gels and by MALDI-TOF analysis of tryptic peptides.

Animals↗
PubMed2003

Purification and diagnostic utility of a recombinant hepatitis E virus capsid protein expressed in insect larvae.

We report here the expression and purification of a truncated form of the hepatitis E virus ORF2 protein (ORF2delta111/deltaTM), from the fat bodies of Spodoptera litura larvae infected with a recombinant baculovirus. The purified protein migrated as a doublet of approximately 56 kDa on SDS-PAGE and was found to be glycosylated by staining with concanavalin A-linked horseradish peroxidase. The protein was used in a sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to HEV. The results showed complete concordance with those obtained using a commercial kit for the detection of anti-HEV antibodies. Antigen expression in the insect larvae system presents a rapid and low-cost method that obviates the need for expensive tissue culture scale-ups or special equipment.

Animals↗
PubMed2003

Human CYP1A1 allelic variants: baculovirus expression and purification, hydrodynamic, spectral, and catalytical properties and their potency in the formation of all-trans-retinoic acid.

Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CYP1A1.4 (T461N), were expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30-90 nmol (1.8-5.4 mg) spectrally active cytochrome P450 per one liter of culture and purified to electrophoretic homogeneity by Ni-agarose chromatography. The recombinant variants were structurally characterized by UV/Vis, ultracentrifugation, and EPR. Optical and EPR spectra showed all three variants predominantly in high spin state; moreover, EPR indicated changes in the electronic structure of the heme iron of the two mutant variants. Sedimentation equilibrium experiments demonstrated the purified variants in dimeric state in the presence of 0.2% emulgen+0.05% cholate. Higher detergent concentration, the presence of imidazole, and cleavage of the His-tag led to monomerization. Catalytic activity of all purified variants was reconstituted with purified human NADPH-P450 reductase and dilaurylphosphatidylcholine. Enzyme kinetics of ethoxyresorufin O-deethylation revealed similar K(m) ( approximately 0.4 microM) for all variants but slightly different V(max) values (CYP1A1.1: 4.2, CYP1A1.2: 7.0, and CYP1A1.4: 3.0 nmol/min/nmol CYP1A1). The extended C-terminus influenced the enzymatic activity only slightly. All three variants are able to produce significant amounts of all-trans-retinoic acid from all-trans-retinal with V(max) of 4.0, 3.3, and 5.6 nmol/min/nmol CYP1A1 and K(m) values of 111, 83, and 250 microM for CYP1A1.1, CYP1A1.2, and CYP1A1.4, respectively. Availability of the three purified human CYP1A1 variants should facilitate further characterization of their role in metabolism of endogenous and exogenous compounds as well as structural studies.

Alleles↗
PubMed2003

Expression and in vitro activation of Manduca sexta prophenoloxidase-activating proteinase-2 precursor (proPAP-2) from baculovirus-infected insect cells.

Prophenoloxidase activation is a component of the immune system in insects and crustaceans. We recently purified and cloned a new prophenoloxidase-activating proteinase (PAP-2) from hemolymph of the tobacco hornworm Manduca sexta [J. Biol. Chem. 278, 3552-3561]. As the terminal component of a putative serine proteinase cascade, this enzyme activates prophenoloxidase (proPO) via limited proteolysis. To purify and study the activating proteinase for PAP-2 from this insect, we expressed the zymogen of PAP-2 (proPAP-2) in insect cells infected by a recombinant baculovirus that harbors the cDNA. To facilitate the purification of proPAP-2, we modified a commercial vector (pFastBac1) by inserting a synthetic DNA fragment encoding a hexahistidine sequence, allowing fusion of the affinity tag to the carboxyl terminus of a protein. After Spodoptera frugiperda Sf21 cells were infected by the virus, recombinant proPAP-2 was efficiently secreted into the media at a concentration of 5.9 microg/ml under the optimal conditions. After ammonium sulfate precipitation, the proenzyme was purified to near homogeneity by affinity chromatography on Ni(2+)-NTA agarose. Western blot analysis indicated that the recombinant proPAP-2 has a mobility slightly lower than that of the zymogen from M. sexta hemolymph. The molecular mass and isoelectric point of proPAP-2 were determined to be 47,573+/-11Da and 6.6, respectively. After the purified proenzyme was added to hemolymph from induced M. sexta larvae, it was rapidly activated by an unknown proteinase in the presence of peptidoglycan.

Amidohydrolases↗
PubMed1996

Expression and characterization of a truncated, soluble, low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor mediates the clearance of apolipoprotein B- and E-containing lipoproteins from the bloodstream. In the current study, we characterized the binding properties of the amino terminus of the LDL receptor. We produced a recombinant baculovirus that encoded the first 354 amino acids, including the endogenous signal sequence, of the human LDL receptor. This truncated receptor protein (LDL-R354) was secreted from recombinant baculovirus-infected Spodoptera frugiperda (Sf-21) cells. Upon electrophoresis, LDL-R354 migrated with a mobility of 55,000. Treatment of cells with tunicamycin decreased the size of the truncated receptor, suggesting the presence of asparagine-linked carbohydrates. Nonreducing SDS-PAGE resulted in at least three discernible bands with M(r)s consistent with the truncated receptor existing as monomers and multimers, suggesting the possibility of intermolecular disulfide cross-linking. All forms of the truncated receptor bound LDL on a ligand blot in a calcium-dependent manner. The purified truncated receptor bound 125I-LDL with high affinity in a competitive binding assay (IC50 = 0.8 microgram/ml). LDL-R354 also bound calcium. This interaction was sensitive to the conformation of the ligand binding domain, as reduction of the disulfide bonds greatly decreased the affinity of the receptor for calcium. The ligand and calcium binding activities of this truncated receptor protein demonstrate that the ligand binding domain of the LDL receptor can fold into a functionally active protein.

Animals↗
PubMed1995

Molecular structure and function of CD4 on murine egg plasma membrane.

In the present study, the expression of the CD4 molecule on murine egg plasma membrane was confirmed by the indirect immunofluorescence (IIF) method. The full-length CD4 cDNA from murine eggs was synthesised by the reverse transcriptase-polymerase chain reaction (RT-PCR) method and its authenticity verified by Southern blot hybridisation using an end-labelled internal oligonucleotide. The results of DNA sequencing showed that the nucleotide sequence of the cDNA of CD4 from murine egg mRNA was identical to that of immune T cells. To demonstrate the direct interaction of CD4 from murine egg with murine sperm cells bearing MHC (major histocompatibility complex) class II molecule, we employed a baculovirus expression system to generate CD4 on the surface of Spodoptera frugiperda (Sf9) cells. Expression of CD4 on Sf9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV)-CD4 was demonstrated by IIF and immunoblotting. The CD4-expressing Sf9 cells adhered to MHC class II-bearing sperm cells since the adhesion was specifically blocked by anti-CD4 monoclonal antibody (mAb) or anti-monomorphic region of MHC class II mAb. Taking our previous and present experimental results together, they strongly suggest that intercellular membrane adhesion between two gametes at the fusion step in fertilisation is mediated by the MHC class II molecule located on the posterior region of the sperm head and the CD4 molecule on egg plasma membrane.

Animals↗
PubMed1997

Expression of Fas-Fas ligand system associated with atresia in murine ovary.

We detected that Fas receptor (Fas) was expressed at transcriptional levels in oocytes/eggs and some granulosa cells of murine ovary, whereas, Fas ligand (FasL) was found to be strongly expressed in granulosa cells by means of in situ hybridisation. These results were supported by an indirect immunofluorescence (IIF) test with anti-Fas monoclonal antibody (mAb)/FasL Ab. The lysates from granulosa cells were precipitated by anti-FasL Ab, exhibiting a specific band at 40 kDa. When zona pellucida (ZP)-free eggs were incubated with granulosa cells in vitro, apoptosis (DNA fragmentation) was induced in the eggs, as detected by the terminal deoxynucleotide transferase mediated dUTP-nick end labelling (TUNEL) method. To examine whether FasL-bearing cells can induce apoptosis in eggs expressing Fas through molecular interaction between FasL and Fas, we employed a baculovirus expression system to generate FasL on the surface of Spodoptera frugiperda (Sf9) cells. The co-incubation of eggs with Autographa californica nuclear polyhedrosis virus (AcNPV) and FasL transfected Sf9 (Sf9-FasL) cells in vitro was performed and resulted in the induction of apoptosis in eggs as detected by the TUNEL method. Apoptosis was absent in eggs co-incubated with AcNPV-1393 transfected Sf9 (Sf9-1393) cells. Thus, ovarian atresia was suggested to be induced by molecular interaction between FasL on granulosa cells and Fas on oocytes/eggs during oogenesis.

Animals↗
PubMed1998

Aberrant expression and dysfunction of Fas antigen in MRL/MpJ-lpr/lpr murine ovary.

In lpr mice the insertion of an early transposable element (ETn) into intron 2 of the Fas gene, which mediates apoptosis, causes the development of massive lymphadenopathy, splenomegaly and autoimmune disease. In the present study we investigated the influence of this mutation on ovarian development of lpr mice. By means of in situ hybridisation, the expression of Fas mRNA was detected at the same levels in the ovarian cells of MRL/MpJ-lpr/lpr (MRL/lpr) mice as in those of MRL/MpJ-(+)/+ (MRL/+) mice. However, indirect immunofluorescence (IIF) staining with anti-Fas monoclonal antibody (mAb) on the membrane of follicle and egg of MRL/lpr mice was significantly weaker than that of MRL/+ mice. Furthermore, the expression level of Fas protein at the 45 kDa band from ovarian cell lysates of MRL/lpr mice was much lower than that of MRL/+ mice. The co-incubation of Spodoptera frugiperda (Sf9)-Fas lig- and (L) cells with eggs of MRL/+ mice resulted in apoptosis of eggs, as detected by the terminal deoxynucleotide transferase mediated dUPT-nick end labelled (TUNEL) method. In contrast the co-incubation of Sf9-FasL cells with eggs of MRL/lpr mice did not generate apoptosis in eggs. Following intraperitoneal administration of anti-Fas mAb into both types of mice, most oocytes, a proportion of granulosa cells in the ovary and hepatocytes in liver of MRL/+ mice were positively stained by the TUNEL method, corresponding to the appearance of DNA fragmented ladders by DNA fragmentation assay, while negative signals were obtained in those cells of MRL/lpr mice. As the mice aged, the ovarian size of MRL/lpr mice was found to be much larger than that of MRL/+ mice due to the increased number of ovarian follicles. Therefore, the ovarian adenopathy in MRL/lpr mice was strongly suggested to be caused by the dysfunction of Fas antigen in the ovary.

Animals↗
PubMed1995

Kinetic properties of the alpha 2 beta 1 and alpha 2 beta 2 isozymes of the Na,K-ATPase.

The presence of multiple isoforms of the alpha and beta subunits of the Na,K-ATPase in most mammalian tissues has hindered the understanding of the roles of the individual isoforms in directing Na,K-ATPase function. Expression of the Na,K-ATPase subunits in insect cells using recombinant baculoviruses has proven to be a useful system for the study of Na,K-ATPase function. Using this system, we have expressed the rat Na,K-ATPase alpha 2 beta 1 and alpha 2 beta 2 isoforms in Sf-9 insect cells, a cell line derived from the ovary of the fall armyworm, Spodoptera frugiperda. Both beta 1 and beta 2 isoforms can efficiently assemble with the alpha 2 subunit to produce catalytically competent Na,K-ATPase molecules. The analysis of the kinetic properties of both isozymes showed that alpha 2 beta 1 and alpha 2 beta 2 have equivalent sensitivities to ouabain, and similar turnover numbers and apparent affinities for K+ and ATP. The dependence on Na+, however, differs between the isozymes, with alpha 2 beta 2 displaying a slightly higher apparent affinity for the cation than alpha 2 beta 1. In addition, the even greater kinetic differences between Na,K-ATPase isozymes varying in alpha isoforms may be important in further differentiating the enzyme. Thus, when compared to the rat alpha 1 beta 1 Na,K-ATPase expressed in Sf-9 cells, the alpha 2 beta 1 and alpha 2 beta 2 isozymes have a lower apparent affinity for K+ and a higher affinity for Na+ and ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Purification and properties of a Golgi-derived (alpha 1,2)-mannosidase-I from baculovirus-infected lepidopteran insect cells (IPLB-SF21AE) with preferential activity toward mannose6-N-acetylglucosamine2.

Because the availability and subcellular distribution of processing mannosidases in cells play such powerful roles in determining ultimate structures of glycoconjugates, we desired to identify, characterize, and investigate possible regulation of mannosidases in infected and noninfected lepidopteran insect cells. Since our previous observations that a mannosidase activity that converted Man6GlcNAc2 to Man5GlcNAc2 was enhanced in virus-infected cells, thus providing the necessary intermediate for further processing to complex-type oligosaccharides, we attempted purification of this enzyme. A mannosidase was isolated and purified from membranes, operationally defined as Golgi, of recombinant baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells. The molecular mass of this protein was approximately 63 kDa. Assays performed by measuring the conversion of NaB3H4-reduced Man6GlcNAc2-ol to Man5GlcNAc-[3H]GlcNAc2-ol demonstrated that the mannosidase activity was dependent on the presence of divalent cations, which was optimal for Ca2+ at pH 6.0. Inclusion of 1-deoxymannojirimycin resulted in 50% inhibition at a concentration of 20 microM, whereas swainsonine did not show such inhibition. No activity was observed with p-nitrophenyl alpha-D-mannoside (4 mM) as a substrate. The preferred reduced oligosaccharide substrate was Man6GlcNAc2-ol, with lower activities obtained with Man9GlcNAc2-ol, Man8GlcNAc2-ol, and Man7GlcNAc2-ol. With Man6GlcNAc2-ol as substrate, products smaller than reduced Man5GlcNAc2-ol were not observed. Mannose was also liberated from the glycoprotein, ovalbumin. These properties are consistent with an enzyme classification as a type I (alpha 1,2)-Man6-mannosidase.

Animals↗
PubMed1995

Comparison of the enzymatic properties of the Na,K-ATPase alpha 3 beta 1 and alpha 3 beta 2 isozymes.

The coexpression of multiple isoforms of the alpha and beta subunits of the Na,K-ATPase in mammalian tissues gives rise to the complex molecular heterogeneity that characterizes the Na pump. The expression of the different Na,K-ATPase isoforms in insect cells using recombinant baculoviruses represents a useful system for the analysis of Na,K-ATPase isoform function. In the present study, we use this system to direct the expression of the rat Na,K-ATPase alpha 3 beta 1 and alpha 3 beta 2 in sf-9 cells, a cell line derived from the ovary of the fall armyworm, Spodoptera frugiperda. The association of alpha 3 with either beta 1 or beta 2 results in catalytically competent Na,K-ATPase isozymes. Analysis of the kinetic characteristics of these enzymes demonstrates that the accompanying beta subunit isoform does not drastically affect the properties of the alpha 3 polypeptide. This is evidenced by the similar turnover numbers, apparent affinities for K+ and ATP, and the comparable high sensitivity to ouabain exhibited by both isozymes. The kinetic dependence on Na+, however, is different for both isozymes, with alpha 3 beta 2 displaying a 1.6-fold higher apparent affinity for the cation than alpha 3 beta 1. Comparison with other Na,K-ATPase isozymes shows that the apparent Na+ affinity of alpha 3 beta 2 is similar to that of the alpha 1 beta 1 Na pump widely expressed in every tissue; nevertheless, its reactivity toward K+, ATP, and ouabain are characteristic of the alpha 3 isoform. The most pronounced kinetic differences in Na,K-ATPase function are a result of variations in alpha isoform composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Characterization of neuronal nitric oxide synthase and a C415H mutant, purified from a baculovirus overexpression system.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide (.NO). A baculovirus overexpression system has been developed for a constitutive NOS isoform, cloned originally from rat cerebellum (B-NOS). Recombinant virus was used at a multiplicity of infection of 5 to infect Spodoptera frugiperda cells in culture, and NOS was expressed to 10% of the total soluble protein at 48 h postinfection. In order to express catalytically active enzyme, it was necessary to supplement the culture media with hemin. This increased the activity of the enzyme 7-fold. A two column affinity purification was developed for the recombinant enzyme, which gave homogeneous protein that migrated at 150 kDa on a denaturing polyacrylamide gel. A Km for L-arginine was determined to be 2.0 +/- 0.4 microM. As isolated, recombinant B-NOS exhibited a Soret maximum at 402 nm, which shifted to 394 nm in the presence of L-arginine. The Soret maximum of the reduced enzyme in the presence of CO was 444 nm. Initial rate steady-state kinetic analysis of the recombinant B-NOS showed evidence of substrate inhibition by L-arginine, which could also be seen in a partially purified preparation of B-NOS from rat cerebella. This substrate inhibition was not observed with the inducible isoform of NOS, purified from immunostimulated murine macrophages. A C415H mutant was overexpressed and purified using the same conditions established for the wild-type recombinant B-NOS. This C415H mutant exhibited no activity and did not bind heme, providing the first experimental evidence to support previously reported primary amino acid comparisons which suggest that C415 provides the coordinating thiolate to the heme moiety in B-NOS.

Amino Acid Oxidoreductases↗
PubMed2003

Engineering sialic acid synthetic ability into insect cells: identifying metabolic bottlenecks and devising strategies to overcome them.

Previous studies have indicated negligible levels of both sialylation and the precursor N-acetylneuraminic acid (Neu5Ac) in a number of insect cell lines grown in serum-free medium. The overexpression of the human sialic acid 9-phosphate synthase (SAS) in combination with N-acetylmannosamine (ManNAc) feeding has been shown to overcome this limitation. In this study we evaluated the potential bottlenecks in the sialic acid synthesis pathway in a Spodoptera frugiperda (Sf9) insect cell line and devised strategies to overcome them by overexpression of the enzymatic pathway enzymes combined with appropriate substrate feeding. Coexpression of SAS and UDP-GlcNAc 2-epimerase/ManNAc kinase, the bifunctional enzyme initiating sialic acid biosynthesis in mammals, resulted in Neu5Ac synthesis without use of any external media supplementation to demonstrate that Neu5Ac could be generated intracellularly in Sf9 cells using natural metabolic precursors. N-Acetylglucosamine (GlcNAc) feeding in combination with this coexpression resulted in much higher levels of Neu5Ac compared to levels obtained with ManNAc feeding with SAS expression alone. The lower Neu5Ac levels obtained with ManNAc feeding suggested limitations in the transport and phosphorylation of ManNAc. The bottleneck in phosphorylation was likely due to utilization of GlcNAc kinase for phosphorylation of ManNAc in insect cells and was overcome by expression of ManNAc kinase. The transport limitation was addressed by the addition of tetra-O-acetylated ManNAc, which is easily taken up by the cells. An alternative sialic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), could also be generated in insect cells, suggesting the potential for controlling not only the production of sialic acids but also the type of sialic acid generated. The levels of KDN could be increased with virtually no Neu5Ac generation when Sf9 cells were fed excess GlcNAc. The results of these studies may be used to enhance the sialylation of target glycoproteins in insect and other eukaryotic expression systems.

Acetylglucosamine↗
PubMed1996

Wild type and mutant human heart (R)-3-hydroxybutyrate dehydrogenase expressed in insect cells.

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. PC is an allosteric activator that enhances NAD(H) binding to BDH. The enzyme serves as a paradigm to study specific lipid-protein interactions in membranes. Analysis of the primary sequence of BDH, as determined by molecular cloning, predicts that lipid binding and substrate specificity are contributed by the C-terminal third of the protein [Marks, A. R., McIntyre, J. O., Duncan, T. M., Erdjument-Bromage, H., Tempst, P., & Fleischer, S. (1992) J. Biol. Chem. 267, 15459-15463]. The mature form of human heart BDH has now been expressed in catalytically active form in insect cells (Sf9, Spodoptera frugiperda) transfected with BDH-cDNA in baculovirus. Endogenous PC in the insect cells fulfills the lipid requirement for the expressed BDH since enzymatic activity is lost upon digestion with phospholipase A2 and restored selectively by reconstitution with PC vesicles. The K(m)s for NAD+ and (R)-3-hydroxybutyrate (R-HOB) of expressed BDH are similar to those for bovine heart or rat liver BDH in mitochondria. Replacing Cys242 (the only cysteine in the C-terminal domain) with serine by site-directed mutagenesis resulted in a 10-fold increase in K(m) for R-HOB with no change in the K(m) for NAD+, indicating a role for Cys242 in substrate binding. Carboxypeptidase cleavage studies had indicated a requirement of the C-terminal for catalysis and a role in lipid binding [Adami, P., Duncan, T. M., McIntyre, J. O., Carter, C. E., Fu, C., Melin, M., Latruffe, N., & Fleischer, S. (1993) Biochem J. 292, 863-872]. We now show that deletion of twelve C-terminal amino acids to form a truncated BDH mutant results in loss of enzymic function. The expression in Sf 9 cells of the constitutively active full-length mature form of human heart BDH and the first expression and characterization of BDH mutants validate this system for structure-function studies of BDH.

3-Hydroxybutyric Acid↗
PubMed1996

Human bleomycin hydrolase: molecular cloning, sequencing, functional expression, and enzymatic characterization.

We have cloned the cDNA of human bleomycin hydrolase (hBH), a protease which is thought to be involved in the metabolic inactivation of the antineoplastic drug bleomycin. The open reading frame consists of 1365 base pairs and is predicted to encode a 52 kDa protein. The protein shares 40% identity with yeast bleomycin hydrolase and contains the conserved active site residues (Cys, His, Asn) characteristic for cysteine proteases of the papain superfamily. Human bleomycin hydrolase has been functionally expressed in Spodoptera frugiperda Sf9 cells using the Autographa californica nuclear polyhedrosis virus. The 52 kDa recombinant protein forms a hexamer of 310 kDa and acts strictly as an aminopeptidase with a broad substrate specificity. The lack of a leader sequence and its pH optimum at 7.2 suggest a cytosolic/nuclear localization. Human bleomycin hydrolase was detected at low to moderate expression levels in most of the human organs tested. Significantly higher RNA levels have been observed in a variety of tumor cell lines. The human enzyme effectively degrades both forms of bleomycin (A2 and B2) in vitro and could indeed be responsible for the resistance of various tumors to this widely used anticancer drug.

Amino Acid Sequence↗
PubMed1996

Human 25-hydroxyvitamin D3-24-hydroxylase, a multicatalytic enzyme.

Human 25-hydroxyvitamin D-24-hydroxylase has been expressed in Spodoptera frugiperda (Sf21) insect cells using the previously cloned cDNA in baculovirus (AcNPV-P450cc24). The activity of recombinant h-P450cc24 required adrenodoxin, adrenodoxin reductase, and NADPH. Incubation of this reconstituted system with 25-OH-[26,27-(3)H]D3 substrate produced several metabolites that were resolved on a normal-phase cyano HPLC system. These products exactly comigrated with authentic standards for 24-oxo-25-OH-D3, 23(S),25-(OH)2D3, 24(R),25-(OH)2D3, and 24-oxo-23(S),25-(OH)2D3. The soluble proteins from Sf21 cells infected with wild-type baculovirus produced neither 24,25-(OH)2D3 nor any of the other 25-OH-D3 metabolites. The products were isolated and subjected to a normal-phase amino HPLC for further separation, purification, and characterization. Comigration on two HPLC systems, periodate cleavage reactions, and NaBH4 reduction established clearly the identity of these metabolites. Incubation of recombinant h-P450cc24 with 25-OH-[3 alpha-3H]D3 led to the isolation of an additional product that comigrated with 24,25,26,27-tetranor-23-OH-D3. Treatment of putative 24,25,26,27-tetranor-23-OH-[3 alpha-3H]D3 with acetic anhydride changed its migration on amino HPLC to a less polar position, indicating acetylation of a hydroxyl group(s). These data demonstrate conclusively that h-P450cc24 is a multicatalytic enzyme catalyzing most, if not all, of the reactions in the C-24/C-23 pathway of 25-OH-D3 metabolism. It is likely that this enzyme by itself converts 25-OH-D3 and 1,25-(OH)2D3 to one of its final excretion products.

Animals↗
PubMed1996

Glycosylation, palmitoylation, and localization of the human D2S receptor in baculovirus-infected insect cells.

In order to evaluate the baculovirus expression system as a means for high-yield production of homogeneous D2S receptor, we have expressed various D2S receptor constructs in two Spodoptera frugiperda cell lines, a Trichoplusia ni and a Mammestra brassicae cell line. To improve expression yield, the environment of the polyhedrin gene translational initiation site was retained by fusing the first 12 codons of the polyhedrin gene to the 5'-end of the D2S receptor coding sequence. The pharmacological profile of the expressed D2S receptor was similar to that reported for neuronal D2 receptors. Sf9 and Tn cells were best suited for overexpression, yielding about 2 x 10(6) and 4 x 10(6) receptors/cell, respectively, corresponding to 6 pmol/mg of cell protein in Sf9 cells and 10 pmol/mg of cell protein in Tn cells. We have developed a D2 receptor-specific anti-peptide antibody to study glycosylation, palmitoylation, and localization of the heterologously produced receptor. Immunoprecipitation of digitonin/cholate-solubilized receptor from control and tunicamycin-treated Sf9, Tn, and Mb cells revealed an apparent molecular mass of 47-48 kDa for the glycosylated receptor and of 39-40 kDa for the unglycosylated receptor. Although pulse-chase studies showed that glycosylation occurred rapidly and efficiently, the glycosylated receptor only constituted a small fraction of the overall produced receptor protein, which was mainly located intracellularly. The glycosylation of the receptor was of the high-mannose-type in contrast to the complex-type glycosylation found in native tissue. The glycosylated D2S receptor was palmitoylated. Glycosylation, however, was not a prerequisite for palmitoylation which was insensitive to tunicamycin, brefeldin A, and monensin. NH2-terminal addition of the signal sequence of prepromelittin to the D2S receptor increased expression levels 2-3-fold and significantly enhanced membrane insertion and processing, resulting in increased targeting of the synthesized receptor to the plasma membrane.

Animals↗
PubMed1997

Differential effects of subunit interactions on protein kinase A- and C-mediated phosphorylation of L-type calcium channels.

We have expressed the pore-forming alpha1S (skeletal muscle isoform) and alpha1C (cardiac/brain isoform) subunits, as well as the accessory beta2a (cardiac/brain isoform) and alpha2/delta subunits of the L-type, dihydropyridine-sensitive calcium (Ca) channels in Spodoptera frugiperda insect cells (Sf9 cells) by infection with recombinant baculoviruses in order to facilitate biochemical studies of these rare, heteromultimeric membrane proteins. Since the L-type channels are believed to be regulated by protein phosphorylation, this expression system allowed us to investigate which subunits could act as substrates for protein kinase A and C (PKA and PKC) and to determine the potential role of subunit interactions in phosphorylation of the channel proteins. Using purified protein kinases in vitro, the membrane-associated alpha1S, alpha1C, and beta2a subunits were demonstrated to be phosphorylated stoichiometrically by PKA. The extent of phosphorylation of these subunits by PKA was similar whether the subunits were expressed alone or in combination. In addition, the alpha1C and beta2a subunits were phosphorylated stoichiometrically by PKC when expressed individually. In contrast, the alpha1S subunit, when expressed alone, was a poor substrate for PKC, despite the fact that this subunit has been shown to be an excellent substrate for PKC in native skeletal muscle membranes. Interestingly, co-expression of alpha1S with the beta2a subunit restored the ability of the alpha1S subunit to serve as a substrate for PKC. These results strongly suggests that subunit interactions play an important and potentially differential role in channel regulation by PKC, whereas phosphorylation of the same subunit by PKA occurs independent of subunit interaction. Furthermore, our results provide biochemical evidence that, when co-expressed, the alpha1C, alpha1S, and beta2a subunits of L-type Ca2+ channels are excellent substrates for PKA and PKC and support the hypothesis that phosphorylation of each of these subunits may participate in channel regulation by these kinases.

Animals↗