Structure of mouse cytosolic sulfotransferase SULT2A8 provides insight into sulfonation of 7α-hydroxyl bile acids.

Cytosolic sulfotransferases (SULTs) catalyze the transfer of a sulfonate group from the cofactor 3'-phosphoadenosine 5'-phosphosulfate to a hydroxyl (OH) containing substrate and play a critical role in the homeostasis of endogenous compounds, including hormones, neurotransmitters, and bile acids. In human, SULT2A1 sulfonates the 3-OH of bile acids; however, bile acid metabolism in mouse is dependent on a 7α-OH sulfonating SULT2A8 via unknown molecular mechanisms. In this study, the crystal structure of SULT2A8 in complex with adenosine 3',5'-diphosphate and cholic acid was resolved at a resolution of 2.5 Å. Structural comparison with human SULT2A1 reveals different conformations of substrate binding loops. In addition, SULT2A8 possesses a unique substrate binding mode that positions the target 7α-OH of the bile acid close to the catalytic site. Furthermore, mapping of the critical residues by mutagenesis and enzyme activity assays further highlighted the importance of Lys44 and His48 for enzyme catalysis and Glu237 in loop 3 on substrate binding and stabilization. In addition, limited proteolysis and thermal shift assays suggested that the cofactor and substrates have protective roles in stabilizing SULT2A8 protein. Together, the findings unveil the structural basis of bile acid sulfonation targeting 7α-OH and shed light on the functional diversity of bile acid metabolism across species.

Male-biased fasting-induced changes in hepatic tauro-cholic acid and plasma cholesterol in Sult2a8-haplodeficient mice.

SULT2A8 is a male-predominant and liver-specific mouse cytosolic sulfotransferase (SULT) that sulfonates 7α-hydroxyl (7α-OH) bile acids in vitro. Sulfonation regulates bile acid homeostasis, which in turn regulates cholesterol and energy metabolism. Using the Sult2a8-heterozygous (HT) mouse model created earlier in our laboratory, we aimed to investigate the physiological role of SULT2A8 in sulfonating 7α-OH bile acids and its impact on energy metabolism in vivo under both fed and energy-deprivation conditions. Disruption of one allele of the Sult2a8 gene in male HT mice resulted in losing ~ 50% of the 7α-OH sulfonating activity compared to wild-type (WT) control, but no significant change in female HT mice. Under the fed condition comparing the levels of hepatic and biliary bile acids as well as plasma/serum energy metabolites, HT mice displayed a profile similar to that of WT mice, suggesting that the Sult2a8-haplodeficient mice conducted normal energy metabolism. However, after 48-h fasting, a significant decrease in plasma cholesterol level was found in male HT mice but without any significant reduction in female HT mice. Of interest, in male Sult2a8-haplodeficient mice, an increase of the hepatic taurine-conjugated cholic acid level was noted but no noticeable change in other tested bile acids after fasting. Taken together, SULT2A8 is a male-specific and key hepatic SULT in metabolizing 7α-OH primary bile acids. During energy deprivation, SULT2A8 is required to maintain the bile acid and cholesterol metabolism, suggesting SULT is a potential therapeutic target for controlling metabolic diseases.

Molecular and functional characterization of tumor-induced factor (TIF): Hamster homolog of CXCL3 (GROγ) displays tumor suppressive activity.

Previously our lab has created a mouse ovarian xenograft model of copy number variation (CNV)-mediated G protein-coupled receptor (GPCR) MAS-driven tumorigenesis, and RNA profiling identified a putative chemokine tumor-induced factor (Tif). Sequence analysis and chemotactic study suggested that Tif was likely to be a hamster homolog of human GROγ (CXCL3) [IJC 125 (2009): 1316-1327]. In the present study, we report the molecular and functional characterization of the Tif gene. Genomic study of CHO-K1 cells indicated that Tif gene consisted of 4 exons, characterized with an antisense B1 element which is embedded in the fourth exon. Two Tif transcripts were identified which shared identical sequences except that a string of 71-nt derived from the antisense B1 element was deficient in the shorter transcript. Of interests, B1-like RNA ladder was detected in xenografts. Functional studies showed that TIF induced chemotaxis and neovessel formation. Pharmacological studies suggested that TIF activated Gi-coupled CXCR2 and induced both calcium mobilization and ERK1/2 phosphorylation, and suppressed forskolin-stimulated cAMP accumulation. In addition, secreted matured TIF functioned as an autocrine factor and promoted anchorage-independent growth. Unexpectedly, TIF delayed the onset of tumor formation, possibly via suppressing proliferation of stromal fibroblasts. However, TIF did not exert any inhibitory effect on tumor growth. Potentially, TIF could be used for preventing cancer relapse.

Identification and characterization of a novel PPARα-regulated and 7α-hydroxyl bile acid-preferring cytosolic sulfotransferase mL-STL (Sult2a8).

PPARα has been known to play a pivotal role in orchestrating lipid, glucose, and amino acid metabolism via transcriptional regulation of its target gene expression during energy deprivation. Recent evidence has also suggested that PPARα is involved in bile acid metabolism, but how PPARα modulates the homeostasis of bile acids during fasting is still not clear. In a mechanistic study aiming to dissect the spectrum of PPARα target genes involved in metabolic response to fasting, we identified a novel mouse gene (herein named mL-STL for mouse liver-sulfotransferase-like) that shared extensive homology with the Sult2a subfamily of a superfamily of cytosolic sulfotransferases, implying its potential function in sulfonation. The mL-STL gene expressed predominantly in liver in fed state, but PPARα was required to sustain its expression during fasting, suggesting a critical role of PPARα in regulating the mL-STL-mediated sulfonation during fasting. Functional studies using recombinant His-tagged mL-STL protein revealed its narrow sulfonating activities toward 7α-hydroxyl primary bile acids, including cholic acid, chenodeoxycholic acid, and α-muricholic acid, and thus suggesting that mL-STL may be the major hepatic bile acid sulfonating enzyme in mice. Together, these studies identified a novel PPARα-dependent gene and uncovered a new role of PPARα as being an essential regulator in bile acid biotransformation via sulfonation during fasting.

Identification and characterization of a novel CXC chemokine in xenograft tumor induced by mas-overexpressing cells.

Overexpressions of G protein-coupled receptor (GPCR) with elevated downstream signaling events have been reported in various tumors. However, the cellular mechanism that GPCR overexpression leads to tumor formation is largely unknown. The orphan GPCR mas was originally isolated from a human epidermoid carcinoma. In vivo studies of mas-overexpressing cells suggested that xenograft tumor formation was positively correlated with the levels of mas expression. Histochemical analysis indicated that xenograft tumor consisted of mas-transfected and stromal cells. Biochemical analyses revealed that cells overexpressing mas exhibited significantly increased anchorage-independent growth, whereas there was no significant difference in cell proliferation in comparison with empty vector-transfected control cells. Expression profiling using mRNA differential display and Northern analysis indicated an elevated expression of GRO and a novel CXC chemokines, tumor-induced factor (TIF), in mas-transfected cells and xenograft tumor. Bacterially expressed recombinant TIF was found to act as a neutrophil chemoattractant in a chemotactic assay. These results suggest that mas overexpression enables anchorage-independent growth of transformed cells, and interplays of secreted chemokines with stromal cells modulate xenograft tumor formation. Importantly, a novel CXC chemokine, TIF, was identified in the xenograft tumor tissues.

One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector.

Millions of candidate clones are commonly obtained following rounds of phage-displayed antibody library panning, and expression of those selected single-chain variable fragment (scFv) is required for secondary functional screening to identify positive clones. Large scale functional screening is often hampered by the time-consuming and labor-intensive subcloning of those candidate scFv clones into a bacterial expression vector carrying an affinity tag for scFv purification and detection. To overcome the limitations and to develop a multiplex approach, an improved hexahistidine tag phagemid vector was constructed for one-step scFv expression and purification. By using hexahistidine as an affinity tag, soluble scFvs can be rapidly and cost-effectively captured from Escherichia coli periplasmic extracts. For proof-of-concept, feasibility of the improved phagemid vector was examined against two scFvs, L17E4d targeting a cell surface antigen and L18Hh5 recognizing a monoclonal antibody (mAb). Using 1 ml of Ni-NTA agarose, 0.2-0.5 mg of soluble scFv was obtained from 1 L of bacteria culture, and the purified scFvs bound specifically to their target antigens with high affinity. Moreover, using two randomly selected hapten-specific scFv phage clones, it was demonstrated that the display of scFvs on phage surface was not affected by the hexahistidine affinity tag. These results suggest the improved phagemid vector allows the shuttle of phage-displayed antibody library panning and functional scFv production. Importantly, the improved phagemid vector can be easily adapted for multiplex screening.

Efficient expression of foreign genes in CHO DHFR(-) cells by electroporation.

DHFR-deficient Chinese hamster ovary (CHO DHFR(-)) cells are the most popular mammalian expression system for inducible amplification of transgene. In order to obtain more stable transfected CHO DHFR(-) cell clones, transfection efficiency of electroporation under different conditions were systemically investigated using plasmid pSV-beta-Gal as reporter gene. Transfection efficiency was proportionally increased with pulse duration and number of pulse applied. In addition, higher transfection efficiency was found in high salt extracellular solution (Berg's and Hank's buffers) than in intracellular solution (cytomix buffer) under the same electroporation condition. The highest transfection efficiency in examined conditions was about 1 in 350 cells (or 0.289%) when cells were electroporated with twice pulses at 400V, 375microF. The present study offers an optimized guideline for introducing exogenous DNA into CHO DHFR(-) cells by electroporation.

The constitutive activity of ghrelin receptors is decreased by co-expression with vasoactive prostanoid receptors when over-expressed in human embryonic kidney 293 cells.

The functional activity of G protein-coupled receptors can be modified by their ability to form oligomeric complexes with G protein-coupled receptors from other receptor families. Emerging evidence suggests that the appetite-regulating hormone ghrelin is a directly acting vasodilator peptide with anti-inflammatory activity, therefore, we have examined the ability of ghrelin receptors to oligomerize with members of the prostanoid receptor family which are also involved in modulating vascular activity and inflammatory responses. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the ability of ghrelin receptors to hetero-oligomerize with prostaglandin E2 receptor subtype EP3-I, prostacyclin receptors, and thromboxane A2 (TPalpha) receptors, when transiently over-expressed in human embryonic kidney 293 cells. These results suggest that hetero-oligomeric interactions between ghrelin receptors and prostanoid receptors are likely to be of biological relevance. Co-transfection of cells with ghrelin receptor and prostanoid receptors significantly decreased ghrelin receptor expression and attenuated its constitutive activation of phospholipase C without changing its affinity for ghrelin. We also observed an increase in the proportion of ghrelin receptors localized intracellularly in the presence of prostanoid receptors. Taken together, these results suggest that the increased expression of prostanoid receptors in conditions of vascular inflammation, such as in atherosclerotic plaques, could influence those cellular responses dependent on the constitutive activation of ghrelin receptors.

Identification and characterization of a novel mouse peroxisome proliferator-activated receptor alpha-regulated and starvation-induced gene, Ppsig.

The peroxisome proliferator-activated receptor alpha (PPARalpha) has been known to play a pivotal role in maintaining the energy balance during fasting; however, the battery of PPARalpha target genes involved in this metabolic response is still not fully characterized. Here, we report the identification and characterization of Ppsig (for PPARalpha-regulated and starvation-induced gene) with unknown biological function from mouse liver. Multiple Ppsig cDNAs which differed in the 3'-untranslated regions were identified. The open reading frame of Ppsig cDNA is 1830 bp which encodes a protein of 67.33 kDa. Ppsig contains 11 exons spanning at least 10 kb. Although the exact biological function of Ppsig is still not known, we found that Ppsig mRNA transcript was dramatically up-regulated during 72 h fasting and following treatment with a potent PPARalpha agonist, in a tissue-specific and PPARalpha-dependent manner. A functional peroxisome proliferator-response element was found in the intron 1 of Ppsig, thus confirming that Ppsig is a novel direct mouse PPARalpha target gene. This finding might help in elucidating the transcriptional regulatory mechanism of Ppsig in the cellular response to fasting.

Dual effect of endothelin 1 on angiotensin II-potentiated purinergic neurotransmission in prostatic rat vas deferens.

Angiotensin and endothelin are vasoactive peptides with neuromodulatory effect, however their interactions in facilitating neurotransmission are largely unknown. In the present study, effort was made to examine how endothelin 1 modulates angiotensin II-potentiated purinergic neurotransmission in prostatic rat vas deferens. Both peptides facilitated field-stimulated muscle contraction in a concentration-dependent manner with Kd values of 16.97+/-6.47 and 2.46+/-0.83 nM for angiotensin II and endothelin 1, respectively. Hill plot analysis gave Hill constants of 0.91+/-0.15 and 0.97+/-0.26 for angiotensin II and endothelin 1, respectively. Correlation analysis indicated that the extent of potentiation by angiotensin II, but not endothelin 1, was proportional to the basal field-stimulated muscle contraction. In the presence of low concentrations of endothelin 1 (< or = 3 nM), angiotensin II-potentiated field-stimulated contraction was further enhanced by endothelin. However, in the presence of high concentrations of endothelin 1 (> or = 10 nM), a much increased basal field-stimulated contraction was observed, and the addition of angiotensin II did not elicit any further enhancement in the contractile response. Intriguingly, after prolonged exposure of prostatic rat vas deferens to a high concentration of endothelin 1, the addition of angiotensin II induced a refractory response to field-stimulation. Taken together, our result indicated that endothelin 1 augmented angiotensin II-facilitated purinergic neurotransmission in prostatic rat vas deferens at low concentrations, but inhibited gradually at high concentrations.

Identification and characterization of surrogate peptide ligand for orphan G protein-coupled receptor mas using phage-displayed peptide library.

In the present study, a phage-displayed random peptide library was used to identify surrogate peptide ligands for orphan GPCR mas. Sequence analysis of the isolated phage clones indicated a selective enrichment of some peptide sequences. Moreover, multiple alignments of the isolated phage clones gave two conserved peptide motifs from which we synthesized peptide MBP7 for further evaluation. Characterization of the representative phage clones and the synthetic peptide MBP7 by immunocytochemistry revealed a strong punctate cell surface staining in CHO cells expressing mas-GFP fusion protein. The isolated phage clones and synthetic peptide MBP7 induced mas internalization in a stable CHO cell clone (MC0M80) over-expressing mas. In addition, MBP7-stimulated phospholipase C activity and intracellular calcium mobilization in these same cells. In summary, we have demonstrated a systematic approach to derive surrogate peptide ligands for orphan GPCRs. With this technique, we have identified two conserved peptide motifs which allow us to identify potential protein partners for mas, and have generated a peptide agonist MBP7 which will be invaluable for functional characterization of the mas oncogene.

A temporal study on the histopathological, biochemical and molecular responses of CCl(4)-induced hepatotoxicity in Cyp2e1-null mice.

Previous study using Cyp2e1-null mice showed that Cyp2e1 is required in CCl(4)-induced liver injury at 24h, what remains unclear are the temporal changes in liver damage and the spectrum of genes involved in this process. We investigated the time-dependent liver changes that occurred at morphological, histopathological, biochemical and molecular levels in both Cyp2e1(+/+) and Cyp2e1(-/-) mice after treating with either corn oil or CCl(4) (1 ml/kg) for 2, 6, 12, 24 and 48 h. A pale orange colored liver, indicative of fatty infiltration, was observed in Cyp2e1(+/+) mice treated with CCl(4) for 24 and 48 h, while the Cyp2e1(+/+) mice treated with corn oil and Cyp2e1(-/-) mice treated with either corn oil or CCl(4) showed normal reddish brown colored liver. Ballooned hepatocytes with multiple vacuoles in their cytoplasm were observed in the livers of Cyp2e1(+/+) mice 24 and 48 h after treating with CCl(4). The levels of serum alanine aminotransferase and aspartate aminotransferase, markers for liver injury, were significantly higher at 12h, peaked at 24h and gradually decreased at 48 h after CCl(4) intoxication. In contrast, this kind of damage was not apparent in the Cyp2e1(-/-) mice treated with CCl(4). Altered expressions of genes related to liver cirrhosis, apoptosis, oxidative stress, xenobiotic detoxification, lipid metabolism, chemsensory signaling or tumorigenesis, structural organization, regeneration and inflammatory response were identified, and the time-dependent changes in expression of these genes were varied. Overall, the present study provides insights into the mechanism of CCl(4)-induced hepatotoxicity in animal models.

Surrogate target cells expressing surface anti-idiotype antibody for the clinical evaluation of an internalizing CD22-specific antibody.

SM03, a chimeric antibody that targets the B-cell restricted antigen CD22, is currently being clinically evaluated for the treatment of lymphomas and other autoimmune diseases in China. SM03 binding to surface CD22 leads to rapid internalization, making the development of an appropriate cell-based bioassay for monitoring changes in SM03 bioactivities during production, purification, storage, and clinical trials difficult. We report herein the development of an anti-idiotype antibody against SM03. Apart from its being used as a surrogate antigen for monitoring SM03 binding affinities, the anti-idiotype antibody was engineered to express as fusion proteins on cell surfaces in a non-internalizing manner, and the engineered cells were used as novel "surrogate target cells" for SM03. SM03-induced complement-mediated cytotoxicity (CMC) against these "surrogate target cells" proved to be an effective bioassay for monitoring changes in Fc functions, including those resulting from minor structural modifications borne within the Fc-appended carbohydrates. The approach can be generally applied for antibodies that target rapidly internalizing or non-surface bound antigens. The combined use of the anti-idiotype antibody and the surrogate target cells could help evaluate clinical parameters associated with safety and efficacies, and possibly the mechanisms of action of SM03.

Application of fluorescent differential display and peroxisome proliferator-activated receptor (PPAR) alpha-null mice to analyze PPAR target genes.

In conclusion, we have applied the fluorescent differential method and the PPAR alpha-null mouse model for the rapid isolation of expression tags of PPAR alpha target genes that are involved in the action of peroxisome proliferators and in the regulation of lipid homeostasis under energy deprivation. Identification of a wide spectrum of PPAR alpha target genes will provide new insights into the diverse cellular pathways regulated by these receptor, and this information will be critical for understanding the complicated biological interactions among members of the PPAR alpha target genes. With the recent technological advancement, a newer method, such as DNA microarray, has emerged in the identification of differential gene expressions. This new DNA microarray method, in conjunction with the differential display method, is the first important step toward understanding the molecular mechanisms of gene interactions in any biological systems and can speed up the search for differential gene expressions.

Immunohistochemical colocalization of type II angiotensin receptors with somatostatin in rat pancreas.

Earlier studies indicate that binding sites of type II angiotensin (AT2) receptors are detected all over the pancreas, as well as in the pancreatic exocrine cell line AR4-2J. However, lack of corresponding functional AT2 receptor responses can be detected in the exocrine pancreas. The aim of present study is to determine the protein expression of AT2 receptors in the pancreas by probing with an AT2 receptor-specific antibody, and to examine the role of AT2 receptors in the regulation of pancreatic endocrine hormone release. In Western protein analysis of adult rat tissues, expression of AT2 receptor-immunoreactive bands of 56, 68, and 78 kDa was detected in the adrenal, kidney, liver, salivary glands, and pancreas. In adult rat pancreas, strong immunoreactivity was detected on cells that were located at the outer region of Langerhans islets. Immunohistochemical studies indicated that AT2 receptors colocalized with somatostatin-producing cells in the endocrine pancreas. Consistent with the findings in adult pancreas, abundant expression of AT2 receptors was also detected in immortalized rat pancreatic endocrinal cells lines RIN-m and RIN-14B. To examine the role of AT2 receptors on somatostatin secretion in the pancreas, angiotensin-stimulated somatostatin release from pancreatic RIN-14B cells was studied by an enzyme immunoassay in the absence or presence of various subtype-selective angiotensin analogues. There was a basal release of somatostatin from RIN-14B cells at a rate of 8.72 +/- 4.21 ng/10(6) cells (n = 7). Angiotensin II (1 nM-10 microM) stimulated a biphasic somatostatin release in a dose-dependent manner with an apparent EC50 value of 49.3 +/- 25.9 nM (n = 5), and reached maximal release at 1 microM angiotensin II (982 +/- 147.34% over basal secretion; n = 5). Moreover, the AT2 receptor-selective angiotensin analogue, CGP42112, was 1000 times more potent than the AT1 receptor-selective angiotensin analogue, losartan, in inhibiting angiotensin II-stimulated somatostatin release. These results suggest that angiotensin may modulate pancreatic hormone release via regulation of somatostatin secretion.

P2Y6 receptor-mediated proinflammatory signaling in human bronchial epithelia.

P2Y receptors are expressed in virtually all epithelia and are responsible for the control of fluid and electrolyte transport. In asthmatic inflammation, the bronchial epithelia are damaged by eosinophil-derived, highly toxic cationic proteins, such as major basic protein (MBP). Consequently, extracellular nucleotides are released into the extracellular space from airway epithelial cells, and act in an autocrine or paracrine fashion to regulate immune functions. Our data show damage to the human bronchial epithelial cell line, 16HBE14o-, by poly-L-arginine-induced UDP release into the extracellular medium. Activation of P2Y6 receptor by its natural ligand, UDP, or its specific agonist, MRS 2693, led to the production of two proinflammatory cytokines, interleukin (IL)-6 and IL-8. This may have resulted from increased IL-6 and IL-8 mRNA expression, and activation of p38 and ERK1/2 MAPK, and NF-κB pathways. Our previous study demonstrated that UDP stimulated transepithelial Cl- secretion via both Ca2+- and cAMP-dependent pathways in 16HBE14o- epithelia. This was further confirmed in this study by simultaneous imaging of Ca2+ and cAMP levels in single cells using the Fura-2 fluorescence technique and a FRET-based approach, respectively. Moreover, the P2Y6 receptor-mediated production of IL-6 and IL-8 was found to be dependent on Ca2+, but not the cAMP/PKA pathway. Together, these studies show that nucleotides released during the airway inflammatory processes will activate P2Y6 receptors, which will lead to further release of inflammatory cytokines. The secretion of cytokines and the formation of such "cytokine networks" play an important role in sustaining the airway inflammatory disease.

Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.

Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines. The nucleolar association of these miRNAs is resistant to various cellular stresses, but highly sensitive to the presence of exogenous nucleic acids. Introduction of both single- and double-stranded DNA as well as double stranded RNA rapidly induce the redistribution of nucleolar miRNAs to the cytoplasm. A similar change in subcellular distribution is also observed in cells infected with the influenza A virus. The partition of miRNAs between the nucleolus and the cytoplasm is affected by Leptomycin B, suggesting a role of Exportin-1 in the intracellular shuttling of miRNAs. This study reveals a previously unknown aspect of miRNA biology, and suggests a possible link between these small noncoding RNAs and the cellular management of foreign genetic materials.

Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes.

Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.