Hydroxylated human homotrimeric collagen I in <i>Agrobacterium tumefaciens</i>‐mediated transient expression and in transgenic tobacco plant

Merle, Christine; Perret, Stéphanie; Lacour, Thomas; Jonval, V.; Hudaverdian, S; Garrone, R.; Ruggiero, Florence; Theisen, Manfred

doi:10.1016/s0014-5793(02)02452-3

Cited by 94 publications

(62 citation statements)

References 17 publications

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“…Recombinant collagen I homotrimer (rColl I) was extracted and purified from fieldgrown tobacco plants transformed with human pro␣1(I) chain cDNA lacking the N-propeptide coding sequence, referred to as PRS⌬Npro␣1(I) (29). Hydroxylated recombinant collagen was obtained by co-transformation of tobacco leaves with the collagen construct PRS⌬Npro␣1(I) and cDNA coding the two subunits of the prolyl-4-hydroxylase, the C. elegans ␣-subunit and the mouse ␤-subunit, as described (31). Collagen concentrations were determined by amino acid analysis.…”

Section: Methodsmentioning

confidence: 99%

Prolyl Hydroxylation of Collagen Type I Is Required for Efficient Binding to Integrin α1β1 and Platelet Glycoprotein VI but Not to α2β1

Perret

Eble

Siljander

et al. 2003

Journal of Biological Chemistry

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Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 . Collagen fibrils also bind to the integrin ␣ 2 ␤ 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO) n , respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptorbinding properties of collagen. We show that ␣ 2 ␤ 1 but not ␣ 1 ␤ 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant ␣ 1 ␤ 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use ␣ 2 ␤ 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by ␣ 1 ␤ 1 . These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.The collagens include the most abundant proteins in mammalian tissues providing a scaffold and framework for extracellular matrix assembly. In addition, they can modulate cell behavior through interactions with cellular receptors. All members of the collagen family are built up of three chains and contain at least one triple helix domain formed by repeating GXY sequences (where X is often proline (P) and Y is often hydroxyproline (O)). Such molecules associate to form complex structures, of which the fibril-forming collagens constitute the most abundant matrix component (1). The higher order organization of the collagens, collagen I being the best example, proved crucial to the triggering of specific cell responses; native triple helical structure is essential for cell and platelet adhesion, and the fibrillar structure is required for platelet activation and aggregation (2).The cell surface receptors ␣ 1 ␤ 1 and ␣ 2 ␤ 1 of the integrin family are principally collagen receptors, although they can interact with other extracellular matrix components (e.g. laminins). The specificity of the more recently discovered collagen binding integrins, ␣ 10 ␤ 1 and ␣ 11 ␤ 1 , remains to be determined. Different receptor families have also been identified as containing collagen receptors, such as the membrane-spanning proteoglycans, syndecans (3), and the recently described tyrosine kinase receptors DDR1 and DDR2 (4). In platelets, the recently described glycoprotein VI is a key signaling collagen receptor and is an immunoglobulinn superfamily receptor (5).Several important clues to the understanding of the interaction of ␣ 2 ␤ 1 integrin with collagen arose from the development of triple helical synthetic peptid...

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Section: Methodsmentioning

confidence: 99%

Prolyl Hydroxylation of Collagen Type I Is Required for Efficient Binding to Integrin α1β1 and Platelet Glycoprotein VI but Not to α2β1

Perret

Eble

Siljander

et al. 2003

Journal of Biological Chemistry

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“…Our aim was to test a transient tobacco-based expression system that allows validation of the production of a given recombinant product in plants in a minimum of time and with a maximum level of ¢delity as compared to results obtained in stable plants. This system has previously been used to validate the expression of antibody molecules [9,29], or to study the possibility to metabolically engineer plant cells to improve the thermal stability of recombinant human collagen [8]. In the latter case, results obtained by transient expression were highly reproducible and predictive for the biochemical quality of protein obtained in stable plants.…”

Section: Discussionmentioning

confidence: 99%

“…Plant tissue was harvested for extraction 4 days after transformation as described in Merle et al [8].…”

Section: Agroin¢ltration Of Tobacco Leavesmentioning

confidence: 99%

“…Our aim was to develop a rapid tobacco-based transient expression system that allows the validation of the best expression targeting hypothesis for a given candidate protein within the shortest possible time and without generation of stable transgenic plants. We have previously shown that an adaptation of the agrobacterium in¢ltration technique [7] allowed us to demonstrate the introduction of a post-translational modi¢cation pathway into tobacco cells by transient coexpression of animal-derived prolyl-hydroxylase and its substrate, collagen, to increase hydroxyprolin content and thereby thermal stability of the collagen triple helix [8]. Vaquero et al have demonstrated with a similar system that di¡erent expression approaches can be tested for recombinant antibodies [9].…”

Section: Introductionmentioning

confidence: 99%

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A transient tobacco expression system coupled to MALDI‐TOF‐MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants

Mokrzycki-Issartel¹,

Bouchon

Farrer³

et al. 2003

FEBS Letters

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Tobacco-based transient expression was employed to elucidate the impact of di¡erential targeting to subcellular compartments on activity and quality of gastric lipase as a model for the production of recombinant glycoproteins in plants. Overall N-linked glycan structures of recombinant lipase were analyzed and for the ¢rst time sugar structures of its four individual N-glycosylation sites were determined in situ by matrix-assisted laser desorption/ionization time-of-£ight mass spectrometry (MALDI-TOF-MS) on a trypsin digest without isolation or deglycosylation of the peptides. Three glycosylation sites contain both complex-type N-glycans and high-mannose-type structures, the fourth is exclusively linked to high-mannose glycans. Although the overall pattern of glycan structures is in£uenced by the targeting, our results show that the type of glycans found linked to a given Asn residue is largely in£uenced by the physico-chemical environment of the site. The transient tobacco system combined with MALDI-TOF-MS appears to be a useful tool for the evaluation of glycoprotein production in plants. ß

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“…Obviously, the correct folding of animal proteins in plants is not a major concern. It has to be noted that in the case of collagen a gene encoding a prolyl-4-hydroxylase (P4H) had to be coexpressed to provide the correct prolyl hydroxylation of the human protein enabling formation of the correct structure (Merle et al, 2002). Although plants do harbor an endogenous P4H, the enzyme does not ensure the correct modification of the repetitive sequence X-Pro-Gly in human collagen.…”

Section: The Secretory Pathway and Proteolytic Cleavagementioning

confidence: 99%

Biopharmaceuticals from Plants: A Multitude of Options for Posttranslational Modifications

Warzecha

2008

Biotechnology and Genetic Engineering Reviews

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In 1982 the first recombinant therapeutic, human insulin, was introduced into the market and started a new branch of pharmaceutical development, manufacture, and therapy options. To date, more than 130 recombinant protein therapeutics have been approved by the US Food and Drug Administration (FDA) and many more are being developed world wide. With the increasing number of protein therapeutics the number of potential production organisms is also expanding, and posttranslational modification of proteins has become a topic of special focus. One major difference between smallmolecule drugs and protein therapeutics is that the latter are reliant on a host organism for their production and this can have a large influence on the final structure and can ultimately affect the pharmacokinetics, immunogenicity, and the function of the protein depending on the production process. Plants can be efficiently used as production systems for recombinant proteins thereby offering a variety of options for transgene targeting and modification. This review is intended to give an overview about the potential of plants to serve as a production system for therapeutic and prophylactic biopharmaceuticals with respect to posttranslational modifications.

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Hydroxylated human homotrimeric collagen I in Agrobacterium tumefaciens‐mediated transient expression and in transgenic tobacco plant

Cited by 94 publications

References 17 publications

Prolyl Hydroxylation of Collagen Type I Is Required for Efficient Binding to Integrin α1β1 and Platelet Glycoprotein VI but Not to α2β1

Prolyl Hydroxylation of Collagen Type I Is Required for Efficient Binding to Integrin α1β1 and Platelet Glycoprotein VI but Not to α2β1

A transient tobacco expression system coupled to MALDI‐TOF‐MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants

Biopharmaceuticals from Plants: A Multitude of Options for Posttranslational Modifications

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