SummaryUsing plants as production factories for therapeutic proteins requires modification of their N -glycosylation pattern because of the immunogenicity of plant-specific sugar residues. In an attempt towards such humanization, we disrupted the genes for α 1,3-fucosyltransferase and β 1,2-xylosyltransferase in Physcomitrella patens by homologous recombination. The single ∆ fuc-t and ∆ xyl-t plants, as well as the double knockout, lacked transcripts of the corresponding genes, but did not differ from the wild-type moss in morphology, growth, development, and ability to secrete a recombinant protein, the human vascular endothelial growth factor VEGF 121 , into the culture medium. N -Glycan analysis, however, revealed the absence of 1,3-fucosyl and / or 1,2-xylosyl residues, respectively. Therefore, the modifications described here represent the key step towards the generation of moss lines suitable for the production of plant-made glycosylated biopharmaceuticals with nonallergenic N -glycans.
The high mobility group (HMG) proteins of the HMGB family are architectural factors in eukaryotic chromatin, which are involved in the regulation of various DNAdependent processes. We have examined the post-translational modifications of five HMGB proteins from maize suspension cultured cells, revealing that HMGB1 and HMGB2/3, but not HMGB4 and HMGB5, are phosphorylated by protein kinase CK2. The phosphorylation sites have been mapped to the acidic C-terminal domains by analysis of tryptic peptides derived from HMGB1 and HMGB2/3 using nanospray ion trap mass spectrometry. In native HMGB1, Ser 149 is constitutively phosphorylated, whereas Ser 133 and Ser 136 are differentially phosphorylated. The functional significance of the CK2-mediated phosphorylation of HMGB proteins was analyzed by circular dichroism measurements showing that the phosphorylation increases the thermal stability of the HMGB proteins. Electrophoretic mobility shift assays demonstrate that the phosphorylation reduces the affinity of the HMGB proteins for linear DNA. The specific recognition of DNA minicircles is not affected by the phosphorylation, but a different pattern of protein-DNA complexes is formed. Collectively, these findings show that phosphorylation of residues within the acidic C-terminal domain of the HMGB proteins can modulate protein stability and the DNA binding properties of the HMGB proteins. High mobility group (HMG)1 proteins represent a heterogeneous class of small and relatively abundant chromatin-associated proteins of eukaryotes (1, 2). Proteins belonging to the subgroup of the HMGB proteins 2 (previously termed HMG1/2 proteins (3)) have in common a distinctive DNA-binding motif, termed the HMG-box domain, in which the global fold is well conserved and consists essentially of three ␣-helices arranged in an L-shape (1, 4). The HMG-box domain mediates both non-sequence-specific binding of these proteins to the minor groove of linear DNA and high affinity interactions with distorted DNA structures such as four-way junctions, minicircles, and cis-platinated DNA (2, 4, 5). In complexes with B DNA, a hydrophobic wedge on the concave surface of the HMG-box domain is inserted into the minor groove of the DNA, which contributes to the extent of DNA bending induced by the protein (5). The DNA interactions of the HMG-box domains, which occur in different plant, vertebrate, insect, and yeast HMGB proteins, are modulated by basic and acidic domains flanking the DNA-binding motif (4). HMGB proteins act as architectural components in chromatin facilitating the assembly of nucleoprotein complexes, which are involved, for instance, in the regulation of transcription and recombination (2, 4).In contrast to other eukaryotes, which usually have two or three different HMGB proteins, (higher) plants contain several HMGB proteins (Ն5 family members). The plant HMGB proteins have a single HMG-box domain, which is flanked by a basic N-terminal domain and an acidic C-terminal domain (6). Although the amino acid sequences of the HMG-box domains o...
SummaryProtein therapeutics represent one of the most increasing areas in the pharmaceutical industry. Plants gain acceptance as attractive alternatives for high-quality and economical protein production. However, as the majority of biopharmaceuticals are glycoproteins, plant-specific N-glycosylation has to be taken into consideration. In Physcomitrella patens (moss), glycoengineering is an applicable tool, and the removal of immunogenic core xylose and fucose residues was realized before. Here, we present the identification of the enzymes that are responsible for terminal glycosylation (a1,4 fucosylation and b1,3 galactosylation) on complex-type N-glycans in moss. The terminal trisaccharide consisting of a1,4 fucose and b1,3 galactose linked to N-acetylglucosamine forms the so-called Lewis A epitope. This epitope is rare on moss wild-type proteins, but was shown to be enriched on complex-type N-glycans of moss-produced recombinant human erythropoietin, while unknown from the native human protein. Via gene targeting of moss galactosyltransferase and fucosyltransferase genes, we identified the gene responsible for terminal glycosylation and were able to completely abolish the formation of Lewis A residues on the recombinant biopharmaceutical.
Recent studies have demonstrated that the reduction of the core fucosylation on N-glycans of human IgGs is responsible for a clearly enhanced antibody-dependent cellular cytotoxicity (ADCC). This finding might give access to improved active therapeutic antibodies. Here, the expression of the tumor antigen-specific antibody IGN311 was performed in a glyco-optimized strain of the moss Physcomitrella patens. Removal of plant specific N-glycan structures in this plant expression host was achieved by targeted knockout of corresponding genes and included quantitative elimination of core fucosylation. Antibodies transiently expressed and secreted by such genetically modified moss protoplasts assembled correctly, showed an unaltered antigen-binding affinity and, in extensive tests, revealed an up to 40-fold enhanced ADCC. Thus, the glyco-engineered moss-based transient expression platform combines a rapid technology with the subsequent analysis of glycooptimized therapeutics with regard to advanced properties.
The structure-specific recognition protein SSRP1 plays a role in transcription and replication in the chromatin context. Mediated by its C-terminal high mobility group (HMG) box domain, SSRP1 binds DNA non-sequence specifically but recognizes certain DNA structures. Using acetic acid urea polyacrylamide gel electrophoresis and mass spectrometry, we have examined the phosphorylation of maize SSRP1 by protein kinase CK2␣. The kinase phosphorylated several amino acid residues in the C-terminal part of the SSRP1 protein.Two phosphorylation sites were mapped in the very Cterminal region next to the HMG box domain, and about seven sites are localized within the acidic domain. Circular dichroism showed that the phosphorylation of the two C-terminal sites by CK2␣ resulted in a structural change in the region of HMG box domain, because the negative peak of the CD spectrum at 222 nm was decreased by ϳ10%. In parallel, the phosphorylation induced the recognition of UV-damaged DNA, whereas the non-phosphorylated protein does not discriminate between UV-damaged DNA and control DNA. The affinity of CK2␣-phosphorylated SSRP1 for the DNA correlates with the degree of UV-induced DNA damage. Moreover, maize SSRP1 can restore the increased UV-sensitivity of a yeast strain lacking the NHP6A/B HMG domain proteins to levels of the control strain. Collectively, these findings indicate a role for SSRP1 in the UV response of eukaryotic cells.In eukaryotes, the packaging of genomic DNA with histones and other proteins into chromatin affects DNA-dependent processes, including transcription, recombination, replication, and repair. In many cases, chromatin represses these processes by restricting the access of DNA binding regulatory factors to their DNA target sites. Numerous nuclear activities have been identified that can overcome the repressive effects of chromatin. Post-translational modification of histones and ATP-dependent, chromatin-remodeling complexes are involved in altering the chromatin structure so that the DNA becomes more accessible to the DNA-binding proteins required for transcription initiation (1, 2). DNA repair and other DNA-dependent processes occurring in the chromatin context are also coupled to the structural properties of chromatin.DNA is frequently damaged by a variety of agents that are of endogenous or environmental origin. For many organisms, the most important environmental mutagen is the ultraviolet component of sunlight. The DNA lesions induced by UV light (mainly dipyrimidine photoproducts) are both mutagenic and cytotoxic. Therefore, constant removal and replacement of damaged nucleotide residues by DNA repair mechanisms is required to maintain the DNA as a carrier of stable genetic information (3, 4). DNA damage and repair processes are influenced to a great extent by the packaging of the DNA into chromatin. The repair machinery is therefore dependent on the assistance of ATP-dependent, chromatin-remodeling complexes (5-7).In human cells, a protein complex termed FACT (facilitates chromatin transcription)...
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