Proteomic profiling of Nguni cattle liver tissue using gel and Gel-Free approaches: methodology development and potential applications

Includes abstract.Includes bibliographical references.In South Africa, resource-poor farmers mainly depend on livestock farming for their livelihoods, with cattle production being the most important livestock sector. As a consequence of natural selection in stressful conditions, Nguni cattle have been reported to be metabolically superior to other cattle breeds under unfavourable conditions. Using proteomics, with mass spectrometry at the core of the analysis, the objective of this study was to establish a reliable set of methods for the protein profiling of Nguni cattle livers. To achieve this several alternative technologies were employed and their outcomes compared namely, two-dimensional electrophoresis, fractionation by solution phase iso-electric focusing-reversed phase chromatography (IEF-RP), offline strong cation exchange- low pH reversed phase chromatography (SCX-RP) and offline high pH reverse phase-low pH reverse phase chromatography (RP-RP). All solution based methods were coupled to a tandem mass spectrometer. Protein identification was performed using the ParagonTMAlgorithm of Protein Pilot v4.0 as well as PEAKS v6. The IEF-RP and RP-RP methods achieved similar results in terms of number of proteins identified. In addition, proteins that play a role in the urea cycle (which is believed to contribute to the Nguni cattle’s enhanced metabolic ability) were all identified with both techniques. The RP-RP method was selected as the most appropriate method for future research linked to this work and will be used in the next phase of this project, on the basis that it is easier to automate compared to the IEF-RP method. It will be used beyond the scope of this work to compare levels of expression and modification of the liver proteins and their isoforms in Nguni and Hereford cattle grown under adverse environmental conditions, in order to identify those that may contribute to enhanced liver metabolism in Nguni cattle. This will be complemented by the identification and characterisation of potential polymorphisms with in such proteins that can be used to select for this trait during breeding

Proteomic profiling of Nguni cattle liver tissue using gel and Gel-Free approaches: methodology development and potential applications

Includes abstract.Includes bibliographical references.In South Africa, resource-poor farmers mainly depend on livestock farming for their livelihoods, with cattle production being the most important livestock sector. As a consequence of natural selection in stressful conditions, Nguni cattle have been reported to be metabolically superior to other cattle breeds under unfavourable conditions. Using proteomics, with mass spectrometry at the core of the analysis, the objective of this study was to establish a reliable set of methods for the protein profiling of Nguni cattle livers. To achieve this several alternative technologies were employed and their outcomes compared namely, two-dimensional electrophoresis, fractionation by solution phase iso-electric focusing-reversed phase chromatography (IEF-RP), offline strong cation exchange- low pH reversed phase chromatography (SCX-RP) and offline high pH reverse phase-low pH reverse phase chromatography (RP-RP). All solution based methods were coupled to a tandem mass spectrometer. Protein identification was performed using the ParagonTMAlgorithm of Protein Pilot v4.0 as well as PEAKS v6. The IEF-RP and RP-RP methods achieved similar results in terms of number of proteins identified. In addition, proteins that play a role in the urea cycle (which is believed to contribute to the Nguni cattle’s enhanced metabolic ability) were all identified with both techniques. The RP-RP method was selected as the most appropriate method for future research linked to this work and will be used in the next phase of this project, on the basis that it is easier to automate compared to the IEF-RP method. It will be used beyond the scope of this work to compare levels of expression and modification of the liver proteins and their isoforms in Nguni and Hereford cattle grown under adverse environmental conditions, in order to identify those that may contribute to enhanced liver metabolism in Nguni cattle. This will be complemented by the identification and characterisation of potential polymorphisms with in such proteins that can be used to select for this trait during breeding

The study of degradation mechanisms of glyco-engineered plant produced anti-rabies monoclonal antibodies E559 and 62-71-3

Rabies is an ancient and neglected zoonotic disease caused by the rabies virus, a neurotropic RNA virus that belongs to the Rhabdoviridae family, genus Lyssavirus. It remains an important public health problem as there are cost and health concerns imposed by the current human post exposure prophylaxis therapy. The use of monoclonal antibodies (mAbs) is therefore an attractive alternative. Rabies mostly affects people that reside in resource-limited areas where there are occasional failures in the cold-chain. These environmental changes may upset the stability of the mAbs. This study focused on mAbs 62-71-3 and E559; their structures, responses to freeze/thaw (F/T) and exposure to reactive oxygen species were therefore studied with the aid of a wide range of biophysical and in silico techniques in order to elucidate their stability and identify aggregation prone regions. E559 was found to be less stable than 62-71-3. The complementarity determining regions (CDR) contributed the most to its instability, more specifically: peptides (EIWD102)-E-99 and (92)ATSPYT(97) found in CDR3, Trp33 found in CDR1 and the oxidised Met34. The constant region "(158)SWNSGALTGHTFPAVL(175)" was also flagged by the special aggregation propensity (SAP) tool and F/T experiments to be highly prone to aggregation. The E559 peptides "(4)LQESGSVL(11) from the heavy chain and (4)LTQSPSSL(11) from the light chain, were also highly affected by F/T. These residues may serve as good candidates for mutation, in the aim to bring forward more stable therapeutic antibodies, thus paving a way to a more safe and efficacious antibody-based cocktail treatment against rabies

Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3.

Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG

Fifty percent end-point neutralisation activity (reciprocal titre) of E559 and 62-71-3 in a modified Rapid Fluorescent Focus Inhibition Test (RFFIT).

<p>Fifty percent end-point neutralisation activity (reciprocal titre) of E559 and 62-71-3 in a modified Rapid Fluorescent Focus Inhibition Test (RFFIT).</p

Far-UV CD spectra of humanised 62-71-3 (black) and E559 (blue) mAbs.

<p>Far-UV CD spectra of humanised 62-71-3 (black) and E559 (blue) mAbs.</p

N-linked glycans on the anti-rabies mAbs.

<p>N-glycosylation profile from E559 HC and LC and from 62-71-3 HC as determined by LC-ESI-MS of glycopeptides obtained upon trypsin digestion. Numbers represent the presence of the different glyco-species in percent of total glycan. The N-glycan nomenclature used was from <a href="http://www.proglycan.com/" target="_blank">www.proglycan.com</a>.</p

Deconvoluted spectrum of intact, reduced 62-71-3 HC.

<p>The inset shows the zoomed-in LC region with theoretical molecular weights for LC and HC indicated. Detected N-linked glycoforms are shown. The N-glycan nomenclature used was from <a href="http://www.proglycan.com/" target="_blank">www.proglycan.com</a>.</p

Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3 - Fig 2

<p><b>Deconvoluted spectrum of intact, reduced E559 LC (A) and intact, reduced E559 HC (B)</b>.Theoretical molecular weights for LC and HC indicated. Detected N-linked glycoforms are shown. The N-glycan nomenclature used was from <a href="http://www.proglycan.com/" target="_blank">www.proglycan.com</a>.</p