Stable Protein Sialylation in Physcomitrella

Abstract

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of β1,4-galactosylated acceptor N-glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

SARS-CoV-2 Antigens Expressed in Plants Detect Antibody Responses in COVID-19 Patients

“…Nonetheless, glycan-specific antibodies may be missed using plant-produced antigens and we may underestimate antibody reactivity in clinical samples. On the other hand, plant glycans, unlike those from mammalian cells, do not contain sialic acid ( Bohlender et al, 2020 ). While the implications of this are not fully elucidated, sialic acid may shield epitopes ( Galili, 2020 ), potentially resulting in increased reactivity to plant-produced proteins.…”

Process engineering of biopharmaceutical production in moss bioreactors via model-based description and evaluation of phytohormone impact

“…Furthermore, down-stream processing (DSP) may be simplified, thus reducing production time and costs (Huang and McDonald, 2009). Among other features, the high rate of homologous recombination observed in the nuclear DNA of somatic cells positioned the moss Physcomitrium patens (Physcomitrella) as an attractive platform for the production of recombinant therapeutic proteins (Decker et al, 2014;Reski et al, 2018;Bohlender et al, 2020).…”

Unexpected arabinosylation after humanization of plant protein N-glycosylation

“…To reach N -glycan sialylation, which has already been stably attained in N. benthamiana and Physcomitrella (Kallolimath et al, 2016; Bohlender et al, 2020), the galactosylated N -glycan acceptor should be provided as a first step. In planta N -glycan β1,4-galactosylation has been achieved via expression of heterologous coding sequences (CDSs) of different versions of β1,4-galactosyltransferases (β1,4-GalT), including the sequences of various animal species along with the human one and chimeric varieties thereof (Palacpac et al, 1999; Bakker et al, 2001, 2006; Misaki et al, 2003; Huether et al, 2005; Fujiyama et al, 2007; Hesselink et al, 2014; Kittur et al, 2020; Kriechbaum et al, 2020).…”

“…In this study we analyzed the galactosylation efficiency of the chimeric β1,4-galactosyltransferase FTGT, which consists of the CTS domain of the moss α1,4-fucosyltransferase fused to the catalytic domain of the human β1,4-GalT (Bohlender et al, 2020), in Physcomitrella expressing human erythropoietin (hEPO) (Weise et al, 2007). Human EPO is a highly glycosylated protein hormone which inhibits apoptosis of erythroid progenitor cells and stimulates their differentiation, increasing the number of circulating mature red blood cells (Jelkmann, 2013).…”

“…However, up to five additional pentoses were found to be attached to about 92% of all β1,4-galactosylated N -glycans. Pentosylation on β1,4-galactosylated N -glycans was recently reported in N. tabacum and Physcomitrella (Bohlender et al, 2020; Kittur et al, 2020), indicating that this modification might affect different plant-based production systems; but so far no reports are available elucidating its identity. Here, we identified the unknown pentoses as α-linked arabinoses.…”