Human Recombinant Type I Collagen Produced in Plants

TISSUE ENGINEERING: Part A Volume 19, Numbers 13 and 14, 2013 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2012.0347

Oded Shoseyov, PhD,1,2 Yehudit Posen, PhD,3 and Frida Grynspan, PhD2

As a central element of the extracellular matrix, collagen is intimately involved in tissue development, remodeling, and repair and confers high tensile strength to tissues. Numerous medical applications, particularly, wound healing, cell therapy, bone reconstruction, and cosmetic technologies, rely on its supportive and healing qualities. Its synthesis and assembly require a multitude of genes and post-translational modifications, where even minor deviations can be deleterious or even fatal. Historically, collagen was always extracted from animal and human cadaver sources, but bare risk of contamination and allergenicity and was subjected to harsh purification conditions resulting in irreversible modifications impeding its biofunctionality. In parallel, the highly complex and stringent post-translational processing of collagen, prerequisite of its viability and proper functioning, sets significant limitations on recombinant expression systems. A tobacco plant expression platform has been recruited to effectively express human collagen, along with three modifying enzymes, critical to collagen maturation. The plant extracted recombinant human collagen type I forms thermally stable helical structures, fibrillates, and demonstrates bioactivity resembling that of native collagen. Deployment of the highly versatile plant-based biofactory can be leveraged toward mass, rapid, and low-cost production of a wide variety of recombinant proteins. As in the case of collagen, proper planning can bypass plant-related limitations, to yield products structurally and functionally identical to their native counterparts.

Collagen Structure and Function lagens are flanked by terminal propeptides, which lack the Gly-Pro-Xaa motif that prevents premature fibrilization. The ollagen, secreted by fibroblasts and epithelial cells, individuality of each collagen type is defined by the final constitutes the most dominant protein of the extracel- C ensemble of domain types. These variances highly influence lular matrix (ECM) and connective tissue, and is intimately the orientation behavior, eventual fibril diameter, and the involved in tissue development, remodeling, repair, and molecular structures formed upon polymerization and their overall physical support. The protein confers high-tensile biomechanical specifications. strength and resilience to tissues, where its profile of post- translational modifications dictates its physical specifications Type I Collagen Biosynthesis and role at each deposition site.1 As the most abundant animal protein, collagen can be found in 27 variations, yet, Orchestration and control of collagen synthesis, trafficking, types I, II, and III make up 80%–90% of bodily collagens.2 fibril assembly, and deposition in the extracellular space re- The soluble, 300 nm long and 1.5 nm wide macromolecule quire a multitude of genes, processing enzymes, associated contains over 300 Gly-X-Y triplet repeats along its chain. proteins, and cofactors (reviewed in Canty and Kadler3 and Upon orderly packing, three collagen polypeptide chains Myllyharju and Kivirikko4). To date, 42 collagen-encoding assemble into a characteristic collagen triple helix to form genes have been identified in the human genome. The three sheet structures or homo- or heterotrimeric fibrils (30–300 nm Type I procollagen polypeptides are encoded by the COL1A1 wide), displaying a staggered pattern at an axial periodicity and COL1A2 genes, located on chromosomes 17q21-22 and of 67 nm. The remarkable abundance of glycine, proline, and 7q21-22, respectively. Their transcription is differentially reg- hydroxyproline, enables a stable coiled-coil conformation of ulated by cytokines, typically of inflammatory nature, medi- the two identical a1 chains together with one a2 chain. Lat- ated by the Janus Kinase family of protein tyrosine kinases eral interactions between chains, in the form of hydrogen and by signal transducers and activators of transcription bonds or aldol cross-links, among others, further stabilize (STATs).5,6 Upon their activation and nuclear translocation, collagen packing and strengthen fibrils. Fibril-forming col- STATs bind to responsive promoter elements, but still require

1The Robert H. Smith Faculty of Agriculture, Food and Environment, The Robert H. Smith Institute of Plant Science and Genetics, The Hebrew University of Jerusalem, Rehovot, Israel. 2CollPlant Ltd., Ness-Ziona, Israel. 3Scintax, Rehovot, Israel.

1527 1528 SHOSEYOV ET AL. the coordinated participation of a range of transcription thology, such as impaired vasculature, tendons, gums, hair, factors.7 Polypeptide collagen chains are synthesized on and skin. The functional relevance of proper collagen triple membrane-bound ribosomes, and they are cotranslationally helix configuration is clinically expressed upon impaired translocated in the endoplasmic reticulum (ER). collagen hydroxylation in scurvy and in osteogenesis imperfecta, associated with severe bone fragility.9,10 Hy- pochondrogenesis, one of the most severe disorders caused Post-Translation Modifications of Procollagen by mutations in COL2A1, is characterized by a short body The highly complex and hierarchical post-translational and limbs, along with abnormal bone formation in the spine processing of collagen is a prerequisite of full maturation and and pelvis. The inherited Ehlers-Danlos type VII connective functionality of the protein and its fibrillar product. The se- tissue disease is caused by inactive ADAMTS2, the enzyme ries of post-translational modifications of procollagen en- responsible for collagen type I, II, and III propeptide exci- ables triple helix nucleation, formation, and protein folding sion.18 The defective collagen molecules accumulate and self- in the ER. Collagen-specific hydroxylases, glycosyl- assemble into abnormal fibrils, which alter tissue strength transferases, proteinases, and one oxidase are required for and structure. the initial modifications of procollagen en route to collagen maturation. Activity of the multienzyme prolyl 4-hydroxy- Formation of Nano- and Micro-Collagen-Based lases (P4H) complex, acting on over 100 proline residues Structures in the Extracellular Space along the collagen polypeptide chain, has been implicated in Decorated collagen fibrils are transported to the plasma directional helical conformation of collagen and in increasing membrane (PM) within Golgi-to-PM transport compart- the protein’s thermal stability2,8 and viability. Upon P4H ments, which then fuse with the PM to form a fibripositor. deficiency or inactivity, collagen secretion is fully prevented Collagen bundles and higher-order structures are assembled and intermediate, partially folded procollagen chains accu- in these extracytoplasmic recesses, where they can linearly mulate within the ER, initiating ER stress and unfolded and laterally expand19 and/or form composite filaments protein responses that contribute to the pathophysiology of with other types of collagen or noncollagenous elements. The inherited ECM disorders.9,10 fibrils then undergo a number of modifying and reinforce- While the extent of lysine hydroxylation among tissue ment processes, including oxidation and cross-linking. Tip- collagens is highly variable, when compared with that of to-tip fusion of collagen fibril intermediates is promoted by proline, it plays a central role in collagen fibrillogenesis and surface-bound small leucine-rich proteoglycans, which in- ECM mineralization.11 Three isoforms of lysyl hydroxylase hibit side-to-side fibrilar aggregation.20 Post-depositional (LH1–3), along with two alternatively spliced LH2 variants, cross-linking and strengthening21 then takes place within the undergo differential expression between tissues and devel- interstitial space. The resulting products preserve an intimate opmental stages and demonstrate specificity to particular association with the cell surface and lay the ground for the domains of the collagen molecules.12 The hydroxylated ly- extensive and complex tissue-specific extracellular network. sine residues act as the single target of glycosylation on collagen molecules, and as sites of oxidative deamination Collagens in Biomedical and Pharmaceutical and covalent cross-linking, critical in fibril stabilization.13 Applications Hydroxylysine glycosylation of the nascent collagen peptide, mediated by b(1-O)galactosyl- and a(1–2) glucosyltransfer- Collagens boast a long history of medical and industrial ase enzymes and found in marked variability among colla- use in healing procedures, tissue reconstruction, and as gens, has been suggested to impact the rate of interaction dermal fillers, where their abundance, biocompatibility, with procollagen proteinases, and the extent of collagen su- biodegradability, and functionality render them ideal scaf- pramolecular aggregation.14 folding materials for human use. Collagen-based scaffolds Peptidyl proline cis-trans isomerase and protein disulfide provide advantageous features for both cell-free and cell- isomerase catalyze intra- and interchain disulfide bonding, in based tissue engineering techniques, defining the space of addition to the role of the former as a b-subunit of collagen tissue ingrowth, and serving as tissue/organ equivalents, P4H, and the role of the latter in triple-helix folding. In ad- respectively. Such scaffolds can be customized to attract dition, the majority of procollagen N and C propeptides are specific cell types or impregnated with factors to be released cleaved by proteases located in transport vesicles, allowing at the graft site. Collagen scaffolds designed to closely mimic for spontaneous self-assembly of collagen fibrils.2 Proangio- natural ECM environs, are widely applied in basic research genic, anti-angiogenic, and chemotaxic functions have been to shed light on cell behavior and disease etiology and assigned to these cleaved, circulating propeptides, depend- pathogenesis.22–25 ing on their sources and on the molecular context in which Specific collagen types are closely associated with stem they accumulate.15,16 and progenitor cell niches and cell clustering,26–28 which play The sequence of events leading up to collagen maturation a critical role in cell self-renewal, differentiation, and func- and assembly must proceed systematically and in its en- tion. These interactions are highly dependent on the triple tirety, to avoid defects in their structure and/or function. The helical GFOGER collagen sequence,29 the protein’s fibrillar stringent structural and post-translational modification re- status, and on integrin binding. These microenvironments quirements of collagens render them highly susceptible to can be reconstituted in vitro, and form the basis of cell ther- mutations; even minor defects in collagen structure or as- apies toward accelerated reconstruction of diseased tis- sembly can have severe clinical consequences due to the sues.30,31 Collagen-based biomaterials in the form of heart protein’s widespread distribution throughout the human valves32 and functional veins33,34 have been particularly body.17 Such abnormalities can be manifested by tissue pa- valuable in cardiac and pulmonary repair ensuing vascular HUMAN RECOMBINANT TYPE I COLLAGEN PRODUCED IN PLANTS 1529 disease-related insult. Similarly, researchers are actively stantthreatofsamplecontaminationbyhostpathogens. pursuing development of bioengineered ligament and ten- High yields of collagen are secreted in the milk of trans- don tissue mimics that can provide mechanical support until genic animals bearing mammary gland-targeting genomic neotissue is established.35 collagen- and P4H-encoding inserts. Yet, the homotrimeric Throughout the multi-stage wound healing process, col- product fails to mimic the native Type I collagen hetero- lagen and collagen-derived fragments provide indispensable trimer and the hypohydroxylated state of the derived col- support for cell aggregation and adhesion, clot formation, lagen resulted in relatively low thermal stability.44 fibroblast recruitment, and adequate scar tissue generation.36 Bacterial, silkworm,45 and yeast expression systems are Thus, biocompatible collagen-based wound dressings con- less expensive, when compared to mammalian cell culture tribute local haemostatic and chemotactic stimuli, while systems, and appropriate for mass production of certain supplying a structural support upon which neotissue can be proteins, yet often lack enzymes and co-factors necessary for formed at enhanced rates. In addition, the highly absorptive proline and lysine hydroxylation and lack of disulfide bridge character of such products accommodates the high exudate formation, rendering them unfit for expression of mature volume characteristic of injured tissue, preserving appro- and functional collagen. While coexpression of collagen and priate moisture balance of the wound bed. Collagen finds mammalian-derived prolyl-hydroxylase in insect cells en- extensive used in cosmetic procedures, in the form of dermal abled the formation of stable, hydroxylated collagen,46 the fillers injected to smooth the overall appearance of the skin ability to effect other critical post-translational modifications or in more aggressive reconstructive surgery. Advancements is still required to ensure a functional mimic of natural col- in fabrication of collagen-based corneae have been made as lagen. Similar coexpression systems in yeast47,48 yielded well37 and may eventually obviate the need for human cor- stable, triple helical collagen, yet, yeast expression systems nea donations. require relatively expensive stainless steel fermentation and support facilities. The advent of plant-made pharmaceutics involving ge- Expression Systems for Production netic manipulations programming plants to express mole- of Recombinant Collagen cules of therapeutic value, has introduced a feasible Historically, collagen used for pharmaceutical and med- alternative to conventional, fermentation-based expression ical applications was extracted from animal or cadaver models. Plant engineering offers cost-effective, safe, manip- sources. However, the age and physiology of the tissue from ulable, and easily scalable protein yields harvestable after which collagen is harvested significantly impact the bio- culture periods significantly shorter than in other expression physical profile of the extracted collagen, as it is continu- systems.49 The protein synthesis pathways highly conserved

ously subject to postdepositional modifications throughout between plant and eukaryote systems, along with the ab- its lifecycle. Further, tissue-derived collagens inevitably sence of human and animal pathogens in plants add to the contain growth factors and/or cytokines contaminants, attractiveness of this system. Another fundamental advan- which can highly influence matrix–cell interactions and tage lies in the easily harvestable leaves, tubers, and seeds overall tissue remodeling prospects. Moreover, extraction that feature extended shelf lives and can serve as a simple protocols inflict irreversible cross-links within the protein, means of storing recombinant proteins before extraction. jeopardizing the biological and mechanical functions of Expression of recombinant proteins in plants can be further collagen. In addition, growing concerns relating to their enhanced via sorting and targeting recombinant expression allerginicity and risk of prion/pathogen transmission have to subcellular compartments, including the nucleus, cytosol, spurred research in pursuit of alternative solutions.38,39 chloroplast, apoplast, ER (reviewed in Fischer and Emans49), Further accentuation of the rising need for safe biomaterials and plastids.50 alternatives has been provided by a recent FDA publication The versatility of plants as bioreactors is becoming in- calling for prohibition of certain cattle materials in the creasingly apparent when considering the wide range of manufacturing of biologics and medical devices intended plant types engineered to produce a diversity of products, for humans.40 Fish-derived collagens, extensively used in from small proteins51 to complex and large antibodies.52 food, glue, and industrial applications, are highly allergenic Production of edible recombinant proteins and vaccines have and exhibit significantly lower stability and performance at been proposed and executed in potatoes,53 lettuce,54 toma- body temperatures, when compared with vertebrate colla- toes,55 and alfalfa.56 Cultivated plant suspension cells allow gens.41 In addition, recent findings have shown that fish similar protein expression under tightly regulated conditions also harbor prions.42 (reviewed in Fischer et al.57). As a non-food crop with a large The expanding need for collagen and its byproducts in leaf mass and prematurity stage harvesting, exploitation of industrial applications has spurred development of high- tobacco plants as biofactories alleviate both concerns of food throughput extraction and recombinant synthesis and pu- and feed supply contamination and of gene flow. rification techniques, designed to provide pure material While the plant-based expression platform combines resembling its natural form. However, the complex bio- numerous advantages over other systems, lack of or inef- synthesis of collagen, involving a relatively large number of ficient enzymatic support often leads to plant-derived re- enzymes regulating its expression and maturation, imposes combinant molecules void of modifications critical to their considerable demands on recombinant expression systems. half-life and activity.58 In the case of recombinant collagen Mammalian cell lines have been proven effective in induc- expression, plant-derived P4H can generate hydroxypro- tion of procollagen expression and secretion, yet require line-containing proteins, but exhibits relatively loose sub- mass culturing volumes, costly nutrient supplementation, strate sequence specificity in comparison to mammalian and extensive time-to-product periods,43 while under con- P4H.58,59 In addition, amino acid analysis of tobacco- 1530 SHOSEYOV ET AL. expressed human collagen59 demonstrated hydroxylysine both proline and lysine hydroxylated, and O-glycosylated content to be less than 2% of that found in bovine collagen, heterotrimeric, recombinant human procollagen type I has suggesting that endogenic plant LH fails to sufficiently been reported.65 Through a series of crossbreedings, two hydroxylate collagen lysines. Hydroxylated type III colla- human genes encoding recombinant heterotrimeric collagen was successfully expressed in tobacco cells transformed gen type I (rhCOL1) were successfully coexpressed in to- with cDNAs encoding full-length collagen and human P4H bacco plant vacuoles with the human P4H-a P4H-b a and b,60,61 but its hydroxyproline content was 16%–25% and LH3 enzymes. Plants coexpressing all five vacuole- lower than human tissue-derived collagen. Moreover, no targeted proteins generated intact procollagen at yields of details of the hydroxylysine and O-glycosylation content 1 g/kg dry tobacco leaves (Fig. 1). The isolated protein are provided. Plant-specific N-glycosylation, which differs contained heterotrimers only, as demonstrated by colla- from mammalian glycans in their core residues and in the genase treatment, to which homotrimeric collagen is re- full absence of galactose and sialic acid units, presents a sistant. In addition, the hydroxylated proline and lysine major limitation on plant-based expression of mammalian content was within the range of that found in human proteins. It is likely that plant glycans induce immunogenic collagen (7%–10% and 0.7%–1%, respectively). As in nat- responses,62 and that these non-native glycosylation pro- ural processes, underhydroxylated collagen molecules files can affect the functionality of recombinant protein. were degraded and negatively selected in the course of the Several attempts have been made to modify the plant N- enzymatic processing of procollagen to atelocollagen. O- glycan biosynthesis machinery. Stable introduction of the glycosylation was low and was identical to natural levels, human b1,4-galactosyltransferase to tobacco plants, then as indicated by mass spectrometry. The acidic and easily crossbred with plants engineered to express the Mgr-48 extractible plant vacuole provided an ideal intracellular mouse monoclonal IgG1, yielded galactosylated N-glycans subcompartment for procollagen expression. The closed at ratios similar to those achieved in mammalian expression environment protected maturing collagen molecules from systems.63 A strategy devised to knock out the plant b1,2- undesirable N-glycosylations, which sequentially occur on xylosyltransferse and a1,3-fucosyltransferse,64 responsible the cytosolic and luminal faces of the ER.66 Plant-extracted for the immunogenicity of xylose- and fucose-containing rhCOL1 formed thermally stable triple helical structures glycoproteins, provides a platform in which b1,4-galacto- and demonstrated fibril-forming capacities (close to 100%) syltransferse can then be overexpressed to act on recombi- and biofunctionality similar to human tissue-derived col- nant proteins in a noncompetitive manner. lagen supporting attachment and expansion of adult var- Integration of a matrix of distinct plant features toward ious primary human cells normally involved in tissue development of a high output system for expression of repair processes.

FIG. 1. Production of recombinant human collagen in tobacco plants. (A) Engineered tobacco plants are easily grown in large masses under regulated greenhouse conditions. (B) The tobacco plant’s high leaf mass renders it ideal for collection for large-scale protein extraction. (C) Tobacco plants were engineered to express five different genes that include two human collagen type I subunits, along with three human enzymes critical for post-translational modification of procollagen type I. All five proteins were specifically designed to be diverted to the plant vacuole. (D) Procollagen type I is extracted from the transgenic tobacco leaves. (E) The procollagen is purified from the protein extract and processed. (F) Pure human recombinant collagen is produced. Color images available online at www.liebertpub.com/tea HUMAN RECOMBINANT TYPE I COLLAGEN PRODUCED IN PLANTS 1531

Closing Remarks 16. Vincourt, J.B., Etienne, S., Cottet, J., Delaunay, C., Malanda, C.B., Lionneton, F., et al. C-propeptides of procollagens I Deployment of the highly versatile plant-based biofactory alpha 1 and II that differentially accumulate in en- can be leveraged toward mass, rapid, and low-cost produc- chondromas versus chondrosarcomas regulate tumor cell tion of a wide variety of recombinant proteins, high-value survival and migration. Cancer Res 70, 4739, 2010. crops and pharmaceutics. As in the case of collagen, proper 17. Bateman, J.F., Boot-Handford, R.P., and Lamande, S.R. planning can bypass plant-related limitations, to yield Genetic diseases of connective tissues: cellular and extra- products structurally and functionally identical to their na- cellular effects of ECM mutations. Nat Rev Genet 10, 173, tive counterparts. 2009. 18. Colige, A., Nuytinck, L., Hausser, I., van Essen, A.J., Thiry, Disclosure Statement M., Herens, C., et al. 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