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Purification of protein C from canine plasma.

ABSTRACT: BACKGROUND: In order to characterize the functional properties of canine protein C (CnPC), the zymogen needs to be purified from plasma. The goals of this study were (1) to purify protein C from fresh frozen canine plasma by barium chloride and ammonium sulphate precipitation, followed by immunoaffinity chromatography using a monoclonal mouse antibody against human protein C (HPC4) and (2) to characterize this protein's structure. RESULTS: The purified protein contained three glycosylated forms of a heavy chain (~49 kDa) and a glycosylated light chain (~ 25 kDa). Tandem mass spectra of the peptides obtained following trypsin digestion and liquid chromatography identified this protein to be protein C (vitamin K-dependent protein C precursor, gi|62078422) with 100% probability. Three glycosylation sites (Asn139, Asn202, and Asn350) were identified by detection of peptides containing an N-linked glycosylation consensus sequon with a 3-dalton increase in mass following incubation of the protein with PNGase F in 18O-labeled water. Following incubation with Protac (a specific activator of protein C), the heavy chain showed a slight decrease in molecular size and amidolytic activity measured by a synthetic chromogenic substrate containing an amide bond [H-D-(?-carbobenzoxyl)-lysyl-prolyl-arginine-paranitroanilide diacetate salt]. The amidolytic activity was increased by ~303-fold in the final protein preparation compared to that in plasma. The purified protein showed concentration-dependent anti-factor V and anti-factor VIII activities in canine plasma in coagulometric factor assays. CONCLUSIONS: These studies showed that CnPC could be purified from plasma using HPC4 and that this protein showed amidolytic and anti-coagulant properties upon activation with Protac.

SUBMITTER: Wong VM

PROVIDER: S-EPMC4212105 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Purification of protein C from canine plasma.

Wong Valerie M VM Bienzle Dorothee D Hayes M Anthony MA Taylor Paul P Wood R Darren RD

BMC veterinary research 20141018

<h4>Background</h4>In order to characterize the functional properties of canine protein C (CnPC), the zymogen needs to be purified from plasma. The goals of this study were (1) to purify protein C from fresh frozen canine plasma by barium chloride and ammonium sulphate precipitation, followed by immunoaffinity chromatography using a monoclonal mouse antibody against human protein C (HPC4) and (2) to characterize this protein's structure.<h4>Results</h4>The purified protein contained three glycos ...[more]

PMID: 25326145

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Project description:IntroductionThe relationship between epilepsy and cognitive dysfunction has been investigated in canines, and memory impairment was prevalent in dogs with epilepsy. Additionally, canines with epilepsy have greater amyloid-β (Aβ) accumulation and neuronal degeneration than healthy controls. The present study investigated plasma Aβ42 levels and performed proteomic profiling in dogs with refractory epilepsy and healthy dogs.MethodsIn total, eight dogs, including four healthy dogs and four dogs with epilepsy, were included in the study. Blood samples were collected to analyze Aβ42 levels and perform proteomic profiling. Changes in the plasma proteomic profiles of dogs were determined by nano liquid chromatography tandem mass spectrometry.Results and discussionThe plasma Aβ42 level was significantly higher in dogs with epilepsy (99 pg/mL) than in healthy dogs (5.9 pg/mL). In total, 155 proteins were identified, and of these, the expression of 40 proteins was altered in epilepsy. Among these proteins, which are linked to neurodegenerative diseases, 10 (25%) were downregulated in dogs with epilepsy, whereas 12 (30%) were upregulated. The expression of the acute phase proteins haptoglobin and α2-macroglobulin significantly differed between the groups. Complement factor H and ceruloplasmin were only detected in epilepsy dogs, suggesting that neuroinflammation plays a role in epileptic seizures. Gelsolin, which is involved in cellular processes and cytoskeletal organization, was only detected in healthy dogs. Gene Ontology annotation revealed that epilepsy can potentially interfere with biological processes, including cellular processes, localization, and responses to stimuli. Seizures compromised key molecular functions, including catalytic activity, molecular function regulation, and binding. Defense/immunity proteins were most significantly modified during the development of epilepsy. In Kyoto Encyclopedia of Genes and Genomes pathway analysis, complement and coagulation cascades were the most relevant signaling pathways affected by seizures. The findings suggested that haptoglobin, ceruloplasmin, α2-macroglobulin, complement factor H, and gelsolin play roles in canine epilepsy and Aβ levels based on proteomic profiling. These proteins could represent diagnostic biomarkers that, after clinical validation, could be used in veterinary practice as well as proteins relevant to disease response pathways. To determine the precise mechanisms underlying these relationships and their implications in canine epilepsy, additional research is required.

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Purification of protein C from canine plasma.

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Purification of protein C from canine plasma.

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