Grote, V. | Search bioRxiv

bioRxiv2021

Cell-free glycoengineering of the recombinant SARS-CoV-2 spike glycoprotein

The baculovirus-insect cell expression system is readily utilized to produce viral glycoproteins for research as well as for subunit vaccines and vaccine candidates, for instance against SARS-CoV-2 infections. However, the glycoforms of recombinant proteins derived from this expression system are inherently different from mammalian cell-derived glycoforms with mainly complex-type N-glycans attached, and the impact of these differences in protein glycosylation on the immunogenicity is severely underinvestigated. This applies also to the SARS-CoV-2 spike glycoprotein, which is the antigen target of all licensed vaccines and vaccine candidates including virus like particles and subunit vaccines that are variants of the spike protein. Here, we expressed the transmembrane-deleted human {beta}-1,2 N-acetlyglucosamintransferases I and II (MGAT1{triangleup}TM and MGAT2{triangleup}TM) and the {beta}-1,4-galactosyltransferase (GalT{triangleup}TM) in E. coli to in-vitro remodel the N-glycans of a recombinant SARS-CoV-2 spike glycoprotein derived from insect cells. In a cell-free sequential one-pot reaction, fucosylated and afucosylated paucimannose-type N-glycans were converted to complex-type galactosylated N-glycans. In the future, this in-vitro glycoengineering approach can be used to efficiently generate a wide range of N-glycans on antigens considered as vaccine candidates for animal trials and preclinical testing to better characterize the impact of N-glycosylation on immunity and to improve the efficacy of protein subunit vaccines.

synthetic biology↗

bioRxiv2020

The medaka alg2 mutant is a model for hypo-N-glycosylation-associated retinitis pigmentosa

Patients suffering from Congenital Disorders of Glycosylation (CDG) carry mutations in components of the evolutionarily highly conserved protein-glycosylation-machinery. Employing targeted genome editing, we modeled alleles in medaka fish based on a mutation described in an ALG2-index patient. The multisystemic phenotypes in our alg2 model closely resembled the patients syndromes. Molecularly, the mutation results in a reduction of the abundance of N-glycans without altering the profile of glycan structures in fish as well as in patient fibroblasts. This hypo-N-glycosylation impacted on protein abundance in two directions. We discovered a putative compensatory upregulation of the basic glycosylation and glycoprotein processing machinery highlighting the regulatory topology of the network. Conversely, proteins of the retinal phototransduction machinery were massively downregulated in the alg2 model. Those relate to the specific loss of rod photoreceptors that fail to be maintained in the alg2 mutants, a condition known as retinitis pigmentosa. Transient supply of human or medaka alg2 mRNA efficiently rescued the phenotypic spectrum and restored viability demonstrating that our alg2 model delivers key traits for the potential treatment of the disorder.

developmental biology↗