Alastair J. McGinness, Susan A. Brooks, Richard Strasser, Jennifer Schoberer, Verena Kriechbaumer
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引用次数: 0
Abstract
Plant cells are a capable system for producing economically and therapeutically important proteins for a variety of applications, and are considered a safer production system than some existing hosts such as bacteria or yeasts. However, plants do not perform protein modifications in the same manner as mammalian cells do. This can impact on protein functionality for plant‐produced human therapeutics. This obstacle can be overcome by creating a plant‐based system capable of ‘humanising’ proteins of interest resulting in a glycosylation profile of synthetic plant‐produced proteins as it would occur in mammalian systems.For this, the human glycosylation enzymes (HuGEs) involved in N‐linked glycosylation N‐acetylglucosaminyltransferase IV and V (GNTIV and GNTV), β‐1,4‐galactosyltransferase (B4GALT1), and α‐2,6‐sialyltransferase (ST6GAL) were expressed in plant cells. For these enzymes to carry out the stepwise glycosylation functions, they need to localise to late Golgi body cisternae. This was achieved by a protein targeting strategy of replacing the mammalian Golgi targeting domains (Cytoplasmic‐Transmembrane‐Stem (CTS) regions) with plant‐specific ones. Using high‐resolution and dynamic confocal microscopy, we show that GNTIV and GNTV were successfully targeted to the medial‐Golgi cisternae while ST6GAL and B4GALT1 were targeted to trans‐Golgi cisternae.Plant cells are a promising system to produce human therapeutics for example proteins used in enzyme replacement therapies. Plants can provide safer and cheaper alternatives to existing expression systems such as mammalian cell culture, bacteria or yeast. An important factor for the functionality of therapeutic proteins though are protein modifications specific to human cells. However, plants do not perform protein modifications in the same manner as human cells do. Therefore, plant cells need to be genetically modified to mimic human protein modifications patterns. The modification of importance here, is called N‐linked glycosylation and adds specific sugar molecules onto the proteins.Here we show the expression of four human glycosylation enzymes, which are required for N‐linked glycosylation, in plant cells.In addition, as these protein modifications are carried out in cells resembling a factory production line, it is important that the human glycosylation enzymes be placed in the correct cellular compartments and in the correct order. This is carried out in Golgi bodies. Golgi bodies are composed of several defined stacks termed cis‐, medial and trans‐Golgi body stacks. For correct protein function, two of these human glycosylation enzymes need to be placed in the medial‐Golgi attacks and the other two in the trans‐Golgi stacks. Using high‐resolution laser microscopy in live plant cells, we show here that the human glycosylation enzymes are sent within the cells to the correct Golgi body stacks. These are first steps to modify plant cells in order to produce human therapeutics.
期刊介绍:
The Journal of Microscopy is the oldest journal dedicated to the science of microscopy and the only peer-reviewed publication of the Royal Microscopical Society. It publishes papers that report on the very latest developments in microscopy such as advances in microscopy techniques or novel areas of application. The Journal does not seek to publish routine applications of microscopy or specimen preparation even though the submission may otherwise have a high scientific merit.
The scope covers research in the physical and biological sciences and covers imaging methods using light, electrons, X-rays and other radiations as well as atomic force and near field techniques. Interdisciplinary research is welcome. Papers pertaining to microscopy are also welcomed on optical theory, spectroscopy, novel specimen preparation and manipulation methods and image recording, processing and analysis including dynamic analysis of living specimens.
Publication types include full papers, hot topic fast tracked communications and review articles. Authors considering submitting a review article should contact the editorial office first.