Generation and biochemical characterization of glycoPEGylated factor VIIa derivatives

2008 ◽  
Vol 100 (05) ◽  
pp. 920-928 ◽  
Author(s):  
Henrik Østergaard ◽  
Robert J. Bayer ◽  
Matt S. Kalo ◽  
Kyle Kinealy ◽  
Pernille K. Holm ◽  
...  
Keyword(s):  
Half Life ◽  
Biochemical Characterization ◽  
Factor Viia ◽  
Protease Domain ◽  
Limited Effect ◽  
Bleeding Episodes ◽  
Coagulation Pathway ◽  
Inhibitor Patient

SummaryProphylaxis with 2–4 times weekly dosing of factor (F)VIII or FIX is established as an efficacious and safe treatment in haemophilia. Although prophylaxis is not readily available for the inhibitor patient,recent studies have demonstrated a reduction in bleeding episodes in inhibitor patients treated with daily infusions of FVIIa. In order to develop a treatment option comparable to prophylaxis with FVIII or FIX we looked to PEGylation which is an established method for prolonging the circulatory half-life of proteins. However, due to the numerous interactions of FVIIa with the cell surface,TF,FIX and FX there are limited options for unspecific chemical modification of FVIIa without loss of activity. Consequently, we explored the GlycoPEGylation™ technology for selective PEGylation of the two N-glycans in the FVIIa light chain and protease domain to generate seven specifically modified derivatives with PEG groups ranging from 2 to 40 kDa. These derivatives were evaluated in vitro for their ability to interact with small synthetic substrates as well as key molecules relevant to function in the coagulation pathway. The results demonstrate that modification of FVIIa using glycoPEGylation has only a very limited effect on the hydrolysis S-2288 and FX activation. However, the modification does to some extend alter the ability of FVIIa to interact with TF and more importantly, reduces the rate of ATIII inhibition by up to 50% which could allow for an extended active half-life in circulation.

Protein Engineering and in Vitro Characterization of a Factor VIIa Analog with Selective Abrogation of Tissue Factor Binding

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4230-4230
Author(s):  
Prafull S. Gandhi ◽  
Hanne Grøn ◽  
Helle H Petersen ◽  
Stine L Reedtz-Runge ◽  
Ole H. Olsen ◽  
...  
Keyword(s):  
Functional Properties ◽  
Light Chain ◽  
Whole Blood ◽  
Factor Viia ◽  
Similar Data ◽  
Protease Domain ◽  
Binding Ability ◽  
Binding Interface

Abstract Background Recombinant FVIIa (NovoSeven®) is used for the treatment of spontaneous bleeds in haemophilia patients with inhibitory antibodies against FVIII or FIX. Two theories have been proposed to describe the mechanism of action of FVIIa (van't Veer C et. al., (2000) Blood 95:1330-5, Monroe DM et. al., (1998) Blood Coagulation Fibrinolysis 9(S1):S15-S20); a) competition with zymogen FVII to bind to surface exposed TF at site of injury, and b) activation of FX by FVIIa bound to phospholipids exposed on activated platelets. This study aims at generation and characterization of a novel reagent that will provide new insights into the extent of TF influence in vascular injury. Aim To engineer and characterize FVIIa variant with abrogated TF binding ability and to address influence of TF on FVIIa activity in bleeding models (in whole blood clotting). Methods Based on the x-ray crystal structure (PDB: 1DAN), the binding interface between soluble TF (sTF) and FVIIa on the FVIIa light chain was targeted for engineering a new N-glycan. FVIIa variant I69N/F71T was expressed in BHK cells. Majority of the FVIIa variant was additionally glycosylated as assessed by PNGase treatment and SDS-PAGE analysis. Low-levels of non-glycosylated FVIIa variant were removed by passing the FVIIa variant through a sTF column. The resulting FVIIa preparation was homogenous as assessed by HPLC analysis and was characterized as reported earlier (Persson E et al., (2009) FEBS J. 276:3099-109). Results We have successfully introduced a new N-glycan in FVIIa by engineering an N-glycosylation site Asn-X-Ser/Thr in the FVIIa light chain that provides steric hindrance to sTF. WT-FVIIa binds to sTF with Kd of 6 nM, I69N/F71T-FVIIa displays abrogated sTF binding ability up to 10 µM sTF. At higher concentrations, sTF may interact with FVIIa protease domain directly. Proteolytic and amidolytic activity of WT and FVIIa variant are comparable in the absence of sTF, suggesting that the additional N-glycan does not perturb the protease domain. Data from FX activation and ATIII inhibition assays show virtually no effect of sTF on the functional properties of I69N/F71T-FVIIa in presence or absence of phospholipids. Thrombelastography (TEG) assay initiated by kaolin in haemophilia like human whole blood, in the presence of a TF specific inhibitory antibody, revealed no significant differences in clotting time (R-time) and maximum thrombus generation (MTG) for WT and I69N/F71T FVIIa variants. Similar data when TEG assay was initiated by re-lipidated TF (Innovin®) showed marked differences between WT and I69N/F71T FVIIa variants suggesting loss in the TF binding ability of the FVIIa variant. Conclusions Present work demonstrates that, using exclusive mutagenesis strategy, it is possible to engineer FVIIa variant with minimal changes in the primary sequence that displays abrogated TF binding ability but intact amidolytic and proteolytic activity. Functional properties of this variant were characterized by in vitro biochemical and TEG assays. Such a variant should prove useful to delineate involvement of TF in FVIIa activity in bleeding models. Disclosures Gandhi: Novo Nordisk A/S: Employment. Grøn:Novo Nordisk A/S: Employment. Petersen:Novo Nordisk A/S: Employment. Reedtz-Runge:Novo Nordisk A/S: Employment. Olsen:Novo Nordisk A/S: Employment. Østergaard:Novo Nordisk A/S: Employment.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

scholarly journals Intermediate filaments in non-neuronal cells of invertebrates: isolation and biochemical characterization of intermediate filaments from the esophageal epithelium of the mollusc Helix pomatia.

1985 ◽  
Vol 101 (2) ◽  
pp. 427-440 ◽  
Author(s):  
E Bartnik ◽  
M Osborn ◽  
K Weber
Keyword(s):  
Positive Reaction ◽  
Intermediate Filaments ◽  
Biochemical Characterization ◽  
Helix Pomatia ◽  
Molar Ratio ◽  
Negative Stain ◽  
Epithelial Morphology ◽  
Lower Chordate

To screen invertebrate tissues for the possible expression of intermediate filaments (IFs), immunofluorescence microscopy with the monoclonal antibody anti-IFA known to detect all mammalian IF proteins was used (Pruss, R. M., R. Mirsky, M. C. Raff, R. Thorpe, A. J. Dowding, and B. H. Anderton. 1981. Cell, 27:419-428). In a limited survey, the lower chordate Branchiostoma as well as the invertebrates Arenicola, Lumbricus, Ascaris, and Helix pomatia revealed a positive reaction primarily on epithelia and on nerves, whereas certain other invertebrates appeared negative. To assess the nature of the positive reaction, Helix pomatia was used since a variety of epithelia was strongly stained by anti-IFA. Fixation-extraction procedures were developed that preserve in electron micrographs of esophagus impressive arrays of IFs as tonofilament bundles. Fractionation procedures performed on single cell preparations document large meshworks of long and curvilinear IF by negative stain. These structures can be purified. One- and two-dimensional gels show three components, all of which are recognized by anti-IFA in immunoblotting: 66 kD/pl 6.35, 53 kD/pl 6.05, and 52 kD/pl 5.95. The molar ratio between the larger and more basic polypeptide and the sum of the two more acidic forms is close to 1. After solubilization in 8.5 M urea, in vitro filament reconstitution is induced when urea is removed by dialysis against 2-50 mM Tris buffer at pH 7.8. The reconstituted filaments contain all three polypeptides. The results establish firmly the existence of invertebrate IFs outside neurones and demonstrate that the esophagus of Helix pomatia displays IFs which in line with the epithelial morphology of the tissue could be related to keratin IF of vertebrates.

In Vitro Biochemical Characterization of Cytokinesis Actin-Binding Proteins

2016 ◽  
pp. 151-179 ◽  
Author(s):  
Dennis Zimmermann ◽  
Alisha N. Morganthaler ◽  
David R. Kovar ◽  
Cristian Suarez
Keyword(s):  
Binding Proteins ◽  
Biochemical Characterization ◽  
Actin Binding ◽  
Actin Binding Proteins

Biochemical characterization of HIV-1 Rev as a potent activator of casein kinase II in vitro

FEBS Letters ◽  
1998 ◽  
Vol 428 (3) ◽  
pp. 235-240 ◽  
Author(s):  
Kenzo Ohtsuki ◽  
Toshiro Maekawa ◽  
Shigeyoshi Harada ◽  
Atsushi Karino ◽  
Yuko Morikawa ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Hiv 1

scholarly journals Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA

2018 ◽  
Vol 76 (4) ◽  
Author(s):  
Kwok-ho Lam ◽  
Stefan Sikorra ◽  
Jasmin Weisemann ◽  
Hannah Maatsch ◽  
Kay Perry ◽  
...  
Keyword(s):  
Botulinum Neurotoxin ◽  
Biochemical Characterization ◽  
Protease Domain

scholarly journals Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine

2019 ◽  
Vol 166 (1) ◽  
pp. 51-66 ◽  
Author(s):  
Jan M Klenk ◽  
Max-Philipp Fischer ◽  
Paulina Dubiel ◽  
Mahima Sharma ◽  
Benjamin Rowlinson ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Metabolic Pathway ◽  
Biochemical Characterization ◽  
Gene Clusters ◽  
Cytochrome P450 Monooxygenases ◽  
Dihydroxyphenylacetic Acid ◽  
Meta Position ◽  
Redox Partners

AbstractCytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

FIBRINOGEN BIRMINGHAM; A NEW CONGENITAL HETERODIMERIC DYSFIBRINOGENEMIA WITH DEFECTIVE FIBRINOPEPTIDE A RELEASE (Aα 16 Arg→His)

1987 ◽  
Author(s):  
K R Siebenlist ◽  
J T Prchal ◽  
M W Masesson
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Hplc Analysis ◽  
Biochemical Characterization ◽  
Clinical Manifestations ◽  
Severe Bleeding ◽  
Fibrinopeptide A ◽  
History Of ◽  
Bleeding Episodes

Aα 16 Arg→His substitutions are common forms of congenital dysfibrinogenemias. Clinical manifestations range from asymptomatic to moderate hemorrhagic tendencies. Biochemical characterization of one such heterozygotic individual (Fibrinogen Louisville, Galanakis, etal. Ann NY Acad Sci 408:644,1983) indicated that only homodimeric fibrinogen molecules (i.e., containing either normal or abnormal Aα chains) were present. We isolated fibrinogen from the plasma of a 23 year old patient with a history of easy bruising and several recent moderate to severe bleeding episodes. Coagulability with reptilase was 677 (65-70%; n=5) whereas with thrombin (Ha) it approached 100%, depending directly upon the time of incubation with enzyme. HPLC analysis of Ila-induced fibrinopeptide release demonstrated the presence of an abnormal A-peptide (A*), amounting to 50% of the total, which was released more slowly than the normal A-peptide (A). Amino acid analysis of A* demonstrated the absence of Arg and the presence of His. Carboxypeptidase digestion confirmed the structure of A* as Aα 16 Arg-→ His. The clot and the soluble clot liquor resulting from reptilase treatment were separated and each was then further treated with Ilato release A*. HPLC analysis indicated that 31% of the total A* present in the sample was associated with the reptilase clot and 697 remained in the clot liquor. This distribution of A* suggests that Fibrinogen Birmingham, unlike Fibrinogen Louisville, contains heterodimeric molecules that are incorporated into the reptilase clottable fraction. This finding is consistent with a process of random hepatic assembly of dimeric fibrinogen molecules in a heterozygotic individual.

scholarly journals Functional characterization of the rod visual pigment of the echidna (Tachyglossus aculeatus), a basal mammal

2012 ◽  
Vol 29 (4-5) ◽  
pp. 211-217 ◽  
Author(s):  
CONSTANZE BICKELMANN ◽  
JAMES M. MORROW ◽  
JOHANNES MÜLLER ◽  
BELINDA S.W. CHANG
Keyword(s):  
Half Life ◽  
Functional Characterization ◽  
Egg Laying ◽  
Sensory Transduction ◽  
Wild Type ◽  
Functional Variation ◽  
Bovine Rhodopsin ◽  
Tachyglossus Aculeatus

AbstractMonotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λmax of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min−1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min−1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.

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