Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

Arabidopsis CPL4 is an essential C-terminal domain phosphatase that suppresses xenobiotic stress responses.

Eukaryotic gene expression is both promoted and inhibited by the reversible phosphorylation of the C-terminal domain of RNA polymerase II (pol II CTD). More than 20 Arabidopsis genes encode CTD phosphatase homologs, including four CTD phosphatase-like (CPL) family members. Although in vitro CTD phosphatase activity has been established for some CPLs, none have been shown to be involved in the phosphoregulation of pol II in vivo. Here we report that CPL4 is a CTD phosphatase essential for the viability of Arabidopsis thaliana. Mass spectrometry analysis identified the pol II subunits RPB1, RPB2 and RPB3 in the affinity-purified CPL4 complex. CPL4 dephosphorylates both Ser2- and Ser5-PO(4) of the CTD in vitro, with a preference for Ser2-PO(4). Arabidopsis plants overexpressing CPL4 accumulated hypophosphorylated pol II, whereas RNA interference-mediated silencing of CPL4 promoted hyperphosphorylation of pol II. A D128A mutation in the conserved DXDXT motif of the CPL4 catalytic domain resulted in a dominant negative form of CPL4, the overexpression of which inhibited transgene expression in transient assays. Inhibition was abolished by truncation of the phosphoprotein-binding Breast Cancer 1 C-terminal domain of CPL4, suggesting that both catalytic function and protein-protein interaction are essential for CPL4-mediated regulation of gene expression. We were unable to recover a homozygous cpl4 mutant, probably due to the zygotic lethality of this mutation. The reduction in CPL4 levels in CPL4(RNAi) plants increased transcript levels of a suite of herbicide/xenobiotic-responsive genes and improved herbicide tolerance, thus suggesting an additional role for CPL4 as a negative regulator of the xenobiotic detoxification pathway.

Evaluation of anti-Fel d 1 IgY ingredient for pet food on growth performance in kittens.

Introduction: The domestic cat (Felis catus) is one of the most common pets. Worldwide, approximately one in five adults are sensitive to cat allergens. The major cat allergen is the secretoglobulin Fel d 1, which is primarily produced in the salivary and sebaceous glands. Chickens produce IgY antibodies, which are similar in structure to mammalian IgG. When chickens are exposed to Fel d 1, anti-Fel d 1-specific IgY (AFD1) is produced and is naturally concentrated in egg yolk. The aim of this study was to evaluate the tolerability, effects on growth and food consumption, and potential adverse effects of a chicken egg product ingredient containing AFD1 in kittens. Methods: This was a blinded, controlled study. Twenty-seven (27) eight-week old kittens were randomly assigned to three feeding groups containing 0 ppm AFD1 (Group 0), 8 ppm AFD1 (Group 1), and 16 ppm AFD1 (Group 2) for 84 days. Veterinary exams and bloodwork were performed on Day 42 and Day 84, and body weight and body condition score (BCS) were monitored weekly. Results: Throughout the study, there were no signs of nutritional deficiency or adverse clinical events in any of the subjects. Administration of a chicken egg product ingredient containing AFD1 in the diet (whether in coating or combination of coating and top dress) had no significant effect on body weight nor food consumption, and all subjects maintained a healthy Body Condition Score (BCS) throughout the study. Moreover, there were no biologically significant differences in the mean clinical chemistry and hematology parameters. Discussion: This study demonstrated that a diet formulated to contain up to 16 ppm AFD1, included in the coating and the top-dress of dry kitten food, was well tolerated, promoted adequate growth, and exhibited no adverse effects.

Disulfide bond assignment of an IgG1 monoclonal antibody by LC-MS with post-column partial reduction.

Confirmation of the correct disulfide linkage and demonstration of the lack of a significant level of scrambled disulfide bonds are critical to ensure the appropriate folding and structure of recombinant monoclonal antibodies. Currently these are typically achieved by carrying out multiple experiments, most commonly via the comparison of the samples before and after reduction by LC-MS and MS/MS. The data are then analyzed by searching across all the possible disulfide linkages manually or with the aid of computer algorithms. To eliminate the need of multiple experiments and complicated data analysis, a simple LC-MS-based method coupled with post-column partial reduction was developed. Using a recombinant monoclonal IgG1 antibody as an example, this method demonstrates the ability to confirm the correct disulfide linkage and the ability to detect scrambled disulfide bonds from a single experiment with a simple data analysis strategy.

Quantitation of asparagine deamidation by isotope labeling and liquid chromatography coupled with mass spectrometry analysis.

Nonenzymatic asparagine (Asn) deamidation is one of the commonly observed posttranslational modifications of proteins. Recent development of several specific analytical methods has allowed for efficient identification and differentiation of the deamidation products (i.e., isoaspartate [isoAsp] and aspartate [Asp]). Isotope labeling of isoAsp and Asp that are generated during sample preparation by 18O has been developed and can differentiate isoAsp and Asp as analytical artifacts from those present in the samples prior to sample preparation for an accurate quantitation. However, the 18O labeling procedure has a limitation due to the additional incorporation of up to two 18O atoms into the peptide C-terminal carboxyl groups. Variability in the incorporation of 18O atoms into the peptide C-terminal carboxyl groups results in complicated mass spectra and hinders data interpretation. This limitation can be overcome by the dissection of the complicated mass spectra using a calculation method presented in the current study. The multiple-step calculation procedure has been successfully employed to determine the levels of isoAsp and Asp that are present in the sample prior to sample treatment.

Determination of deamidation artifacts introduced by sample preparation using 18O-labeling and tandem mass spectrometry analysis.

The sites and levels of Asn deamidation in proteins are often determined by LC-MS analysis of peptides obtained from enzymatic digestion. However, deamidation that occurs during sample preparation steps results in overestimation of the original level of deamidation. The inherent deamidation and those introduced by sample preparation can be differentiated by preparing samples in (18)O water. When using H(2)(18)O, the formation of isoAsp and Asp by Asn deamidation during sample preparation results in a molecular weight increase of 3 Da due to the incorporation of the (18)O atom to the side chains of isoAsp or Asp; in contrast, inherent deamidation only results in a molecular weight increase of 1 Da. In addition, up to two (18)O atoms can also be incorporated into the peptide C-terminal carboxyl group during enzymatic digestion. Therefore, the 2 Da molecular weight difference at the deamidation sites can only be used to differentiate deamidation that occurs prior to or during sample preparation under conditions that a fixed number of (18)O atoms are incorporated into the peptide C-terminal carboxyl groups. Otherwise, it is challenging to apply this procedure because of the resulting complicated isotopic distributions. Here, a new procedure of using (18)O-water for sample preparation coupled with tandem mass spectrometry (MS/MS) was established to calculate the deamidation artifacts. In this method, b ions were used for the calculation of Asn deamidation that occurred prior to or during sample preparation, which eliminated the complicated factor of various number of (18)O-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions.

Comparison of owner satisfaction between stifle joint orthoses and tibial plateau leveling osteotomy for the management of cranial cruciate ligament disease in dogs.

OBJECTIVE To compare owner satisfaction between custom-made stifle joint orthoses and tibial plateau leveling osteotomy (TPLO) for the management of medium- and large-breed dogs with cranial cruciate ligament disease (CCLD). DESIGN Owner survey. SAMPLE 819 and 203 owners of dogs with CCLD that were managed with a custom-made stifle joint orthosis or TPLO, respectively. PROCEDURES Client databases of an orthosis provider and veterinary teaching hospital were reviewed to identify potential survey respondents. An online survey was developed to evaluate owner-reported outcomes, complications, and satisfaction associated with the nonsurgical (orthosis group) and surgical (TPLO group) interventions. Survey responses were compared between groups. RESULTS The response rate was 25% (203/819) and 37% (76/203) for the orthosis and TPLO groups, respectively. The proportion of owners who reported that their dogs had mild or no lameness and rated the intervention as excellent, very good, or good was significantly greater for the TPLO group than for the orthosis group. However, ≥ 85% of respondents in both groups reported that they would choose the selected treatment again. Of 151 respondents from the orthosis group, 70 (46%) reported skin lesions associated with the device, 16 (11%) reported that the dog subsequently underwent surgery, and 10 (7%) reported that the dog never tolerated the device. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated high owner satisfaction rates for both interventions. Owners considering nonsurgical management with an orthosis should be advised about potential complications such as persistent lameness, skin lesions, patient intolerance of the device, and the need for subsequent surgery.

Sleep apnea in male patients with the fibromyalgia syndrome.

PURPOSE: Fibromyalgia is a common pain syndrome that is often associated with sleep disturbances. The most characteristic pattern noted on formal sleep study is alpha-wave intrusion on delta-wave sleep. This nonrestorative sleep pattern may be endogenous, or caused by any of a number of sleep disturbances. Our goal was to determine the frequency of sleep apnea and its relationship to a nonrestorative sleep pattern in our patients with fibromyalgia syndrome. PATIENTS AND METHODS: All new fibromyalgia patients seen in the Rheumatology Clinic at Fitzsimons Army Medical Center were screened using history and physical examination for suspicion of sleep apnea. When this condition was suspected, the patients underwent formal polysomnography to delineate any sleep disturbance. RESULTS: Four of 92 women, and 13 of 25 men with the new diagnosis of fibromyalgia syndrome underwent polysomnography. Of the women, 2.2% (2 of 92) had significant sleep apnea at formal evaluation; both were obese and had obstructive findings. In contrast, 44% (11 of 25) of the men had significant sleep apnea. CONCLUSIONS: Sleep apnea is not a significant cause of fibromyalgia symptoms in females. In male patients with fibromyalgia, sleep apnea was observed in a large percentage. Fibromyalgia may be a marker for occult sleep apnea in males.

Regulation of abiotic stress signalling by Arabidopsis C-terminal domain phosphatase-like 1 requires interaction with a k-homology domain-containing protein.

Arabidopsis thaliana CARBOXYL-TERMINAL DOMAIN (CTD) PHOSPHATASE-LIKE 1 (CPL1) regulates plant transcriptional responses to diverse stress signals. Unlike typical CTD phosphatases, CPL1 contains two double-stranded (ds) RNA binding motifs (dsRBMs) at its C-terminus. Some dsRBMs can bind to dsRNA and/or other proteins, but the function of the CPL1 dsRBMs has remained obscure. Here, we report identification of REGULATOR OF CBF GENE EXPRESSION 3 (RCF3) as a CPL1-interacting protein. RCF3 co-purified with tandem-affinity-tagged CPL1 from cultured Arabidopsis cells and contains multiple K-homology (KH) domains, which were predicted to be important for binding to single-stranded DNA/RNA. Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation analyses established that CPL1 and RCF3 strongly associate in vivo, an interaction mediated by the dsRBM1 of CPL1 and the KH3/KH4 domains of RCF3. Mapping of functional regions of CPL1 indicated that CPL1 in vivo function requires the dsRBM1, catalytic activity, and nuclear targeting of CPL1. Gene expression profiles of rcf3 and cpl1 mutants were similar during iron deficiency, but were distinct during the cold response. These results suggest that tethering CPL1 to RCF3 via dsRBM1 is part of the mechanism that confers specificity to CPL1-mediated transcriptional regulation.

Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function.

The disulfide bond structures established decades ago for immunoglobulins have been challenged by findings from extensive characterization of recombinant and human monoclonal IgG antibodies. Non-classical disulfide bond structure was first identified in IgG4 and later in IgG2 antibodies. Although, cysteine residues should be in the disulfide bonded states, free sulfhydryls have been detected in all subclasses of IgG antibodies. In addition, disulfide bonds are susceptible to chemical modifications, which can further generate structural variants such as IgG antibodies with trisulfide bond or thioether linkages. Trisulfide bond formation has also been observed for IgG of all subclasses. Degradation of disulfide bond through ß-elimination generates free sulfhydryls disulfide and dehydroalanine. Further reaction between free sulfhydryl and dehydroalanine leads to the formation of a non-reducible cross-linked species. Hydrolysis of the dehydroalanine residue contributes substantially to antibody hinge region fragmentation. The effect of these disulfide bond variations on antibody structure, stability and biological function are discussed in this review.

LC-MS analysis of glycopeptides of recombinant monoclonal antibodies by a rapid digestion procedure.

N-glycan analysis of recombinant monoclonal antibodies (mAbs) usually requires the removal of oligosaccharides by PNGase F followed by 2-AB labeling, normal phase high performance liquid chromatography (NP-HPLC) separation and fluorescence detection. Alternatively antibodies can be completely digested by trypsin to generate glycopeptides for analysis by liquid chromatography-mass spectrometry (LC-MS). Here, we report the development of a rapid digestion procedure to generate glycopeptides for quantitative LC-MS analysis. Recombinant monoclonal antibodies were digested using a combination of Lys-C and trypsin at 37°C for 15 min. The glycan profiles from this rapid digestion procedure are in good agreement with those from LC-MS analysis of glycopeptides from completely digested antibodies and those from NP-HPLC analysis of 2-AB labeled PNGase F released oligosaccharides. This rapid digestion procedure was applied to the comparison of oligosaccharides of two different antibodies. Glycopeptides from the two antibodies were differentially labeled with stable isotopes and analyzed simultaneously after a 1:1 mixing. The combination of the rapid digestion procedure and differential stable isotope labeling significantly reduced the turnaround time.

Detection and quantitation of afucosylated N-linked oligosaccharides in recombinant monoclonal antibodies using enzymatic digestion and LC-MS.

The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the core-fucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.

Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies.

The existence of multiple variants with differences in either charge, molecular weight or other properties is a common feature of monoclonal antibodies. These charge variants are generally referred to as acidic or basic compared with the main species. The chemical nature of the main species is usually well-understood, but understanding the chemical nature of acidic and basic species, and the differences between all three species, is critical for process development and formulation design. Complete understanding of acidic and basic species, however, is challenging because both species are known to contain multiple modifications, and it is likely that more modifications may be discovered. This review focuses on the current understanding of the modifications that can result in the generation of acidic and basic species and their affect on antibody structure, stability and biological functions. Chromatography elution profiles and several critical aspects regarding fraction collection and sample preparations necessary for detailed characterization are also discussed.

Advances in the assessment and control of the effector functions of therapeutic antibodies.

The Fc (crystallizable fragment) region of therapeutic antibodies can have an important role in their safety and efficacy. Although much is known about the structure-activity relationship of antibodies and the factors that influence Fc effector functions, a process has not yet been defined to clearly delineate how Fc functionality should be assessed and controlled during antibody development and manufacturing. In this article, we summarize the current knowledge of antibody Fc functionality, provide a strategy for assessing the effector functions of different classes of therapeutic antibodies (including Fc fusion proteins) and propose a path for routine testing and controls for manufacturers of antibody products.

Vascular endothelial growth factor as a biomarker for the early detection of cancer using a whole cell-based biosensor.

Vascular endothelial growth factor (VEGF) is a cytokine and endothelial cell (EC) mitogen that has been studied for its role in angiogenesis of malignant tumors. Elevated quantities of VEGF in the serum and plasma of patients have been correlated with the presence of cancer and metastasis. Since VEGF induces hyperpermeability of EC monolayers, this protein can be detected in vitro with a whole cell-based biosensor. This biosensor consists of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) attached to a cellulose triacetate (CTA) membrane of an ion-selective electrode (ISE). Previous studies regarding this biosensor have shown that when the biosensor was exposed to a model toxin, such as histamine, the response of the biosensor served as an indirect measurement of the presence of histamine. Similarly, the biosensor responds to the presence of VEGF, but is much more sensitive because VEGF is known to be 50,000-fold more potent than histamine when inducing EC hyperpermeability. The ISE response increased with increasing VEGF concentration. Since lower concentrations required more exposure time, the detection limit was established as a function of exposure time (2-10 h). The practical applicability of the biosensor was also established with cultured human melanoma cells WM793 (nonmetastatic) and 1205LU (metastatic). The resultant change in the potential values revealed significant production of VEGF from the 1205LU cells. A VEGF ELISA was performed to confirm the VEGF concentration in each sample. The biosensor closely predicted the concentrations determined through the ELISA. These results support the use of a cell-based ISE as a quick screening method for the presence of VEGF.

Development of a whole-cell-based biosensor for detecting histamine as a model toxin.

A novel whole-cell potentiometric biosensor for screening of toxins has been developed. The constructed biosensor consists of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) attached to an ion-selective cellulose triacetate (CTA) membrane modified with a covalently attached RGD (arginine-glycine-aspartic acid) peptide sequence. When the HUVECs form a confluent monolayer, ion transport is almost completely inhibited, thereby reducing the response of the ion-selective electrode (ISE). When the monolayer is exposed to agents that increase its permeability (e.g., toxins), ions can diffuse through the membrane, and a potential response from the ISE is achieved. Histamine, a model toxin that increases the permeability of HUVEC monolayers, was used in this study. When the cell-based membranes are exposed to varying concentrations of histamine, the overall response increases with increasing histamine concentration. Thus, the measured potential is an indirect measurement of the histamine concentration. Further experiments were performed for a similar molecule, l-histidine, to test for selectivity. The cell permeability was unaffected by l-histidine, and the sensor response remained unchanged. This type of sensor should find multiple applications in medical, food, and environmental fields and in homeland security.

Experimental evaluation of urinary bladder marsupialization in male goats.

OBJECTIVE: To evaluate the outcome of urinary bladder marsupialization in male goats. STUDY DESIGN: Prospective, experimental study. ANIMALS: Six healthy mixed-breed male goats. METHODS: After experimentally induced urethral obstruction, 6 male goats had urinary bladder marsupialization. Renal ultrasonography, complete blood count, and serum biochemical analysis were evaluated preoperatively (day 0), at 7 postoperative days, and then at 30-day intervals until 180 days. Stomal diameter was recorded immediately postoperatively and at each postoperative interval. Necropsy examination was performed on day 180 or when stomal stricture or death occurred. RESULTS: Stomal stricture occurred in 1 goat at 120 days. Another goat was found dead at 150 days; severe, suppurative cystitis was identified on necropsy. All goats had mild urine scald dermatitis. Serum biochemical values remained within normal limits, but significant decreases in white blood cell count, serum creatinine concentration, and stomal diameter occurred. At necropsy, all bladders were tubular in shape. Histological evidence of chronic suppurative cystitis and chronic, mild lymphoplasmacytic pyelitis occurred in all goats. Bacterial culture of renal tissue yielded growth in 3 goats, and bladder mucosal swabs yielded bacterial growth in all goats. CONCLUSIONS: Although clinical signs of ascending urinary tract infection were not observed in goats with patent stomata, urinary bladder marsupialization may result in ascending urinary tract inflammation or infection. CLINICAL RELEVANCE: Based on our results, urinary bladder marsupialization should be recommended with caution as the primary method for management of urinary tract obstruction in clinical cases.