Strasser, R. | Search bioRxiv

bioRxiv2022

Glyco-engineered pentameric SARS-CoV-2 IgMs show superior activities compared to IgG1 orthologues

Immunoglobulin M (IgM) is the largest antibody isotype with unique features like extensive glycosylation and oligomerization. Major hurdles in characterizing its properties are difficulties in the production of well-defined multimers. Here we report the expression of two SARS-CoV-2 neutralizing monoclonal antibodies in glycoengineered plants. Isotype switch from IgG1 to IgM resulted in the production of pentameric IgMs, comprising of correctly assembled 21 human protein subunits. All four recombinant monoclonal antibodies carried a highly reproducible human-type N-glycosylation profile, with a single dominant N-glycan species at each glycosite. Both pentameric IgMs exhibited increased antigen binding and virus neutralization potency, up to 390-fold, compared to the parental IgG1. Collectively, the results may impact on the future design of vaccines, diagnostics and antibody-based therapies and emphasize the versatile use of plants for the expression of highly complex human proteins with targeted posttranslational modifications.

molecular biology↗

bioRxiv2022

Endoplasmic reticulum calnexins participate in the primary root growth response to phosphate deficiency

Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding (Unfolded Protein Response; UPR). ER stress has been associated with abiotic stress conditions that affect protein folding, including salt stress. However, the role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed several Arabidopsis thaliana mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary roots response to phosphate (Pi) deficiency. CNX and calreticulin (CRT) are homologous ER lectins that bind to N-glycosylated proteins to promote their folding. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. The root growth phenotype was Fe dependent and was associated with Fe accumulation in the root. Two genes involved in Fe-dependent inhibition of root growth under Pi deficiency, the ferroxidase gene LPR1 and P5-type ATPase PDR2, are epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. cnx1-1 cnx2-2 showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency. One sentence summaryCalnexin, a lectin chaperone engaged in the folding of N-glycosylated proteins in the ER, participates in primary root adaptation to low phosphate conditions.

plant biology↗