Suckau, D. | Search bioRxiv

bioRxiv2021

Structural and functional characterization of SARS-CoV-2 RBD domains produced in mammalian cells

As the SARS-CoV-2 pandemic is still ongoing and dramatically influences our life, the need for recombinant proteins for diagnostics, vaccine development, and research is very high. The spike (S) protein, and particularly its receptor binding domain (RBD), mediates the interaction with the ACE2 receptor on host cells and may be modulated by its structural features. Therefore, well characterized recombinant RBDs are essential. We have performed an in-depth structural and functional characterization of RBDs expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells. To structurally characterize the native RBDs (comprising N- and O-glycans and additional posttranslational modifications) a multilevel mass spectrometric approach was employed. Released glycan and glycopeptide analysis were integrated with intact mass analysis, glycan-enzymatic dissection and top-down sequencing for comprehensive annotation of RBD proteoforms. The data showed distinct glycosylation for CHO- and HEK293-RBD with the latter exhibiting antenna fucosylation, higher level of sialylation and a combination of core 1 and core 2 type O-glycans. Additionally, from both putative O-glycosylation sites, we could confirm that O-glycosylation was exclusively present at T323, which was previously unknown. For both RBDs, the binding to SARS-CoV-2 antibodies of positive patients and affinity to ACE2 receptor was addressed showing comparable results. This work not only offers insights into RBD structural and functional features but also provides a workflow for characterization of new RBDs and batch-to-batch comparison.

biochemistry↗

bioRxiv2020

Screening for New Interaction Partners for a Group IIB Intron Ribozyme with Surface Plasmon Resonance Imaging Coupled to MALDI Mass Spectrometry

We coupled SPR imaging (SPRi) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) to identify new potential RNA binders. Here, we improve this powerful method, especially by optimizing the proteolytic digestion (type of reducing agent, its concentration, and incubation time), to work with complex mixtures, specifically a lysate of the rough mitochondrial fraction from yeast. The advantages of this hyphenated method compared to column-based or separate analyses are (i) rapid and direct visual readout from the SPRi array, (ii) possibility of high-throughput analysis of different interactions in parallel, (iii) high sensitivity, and (iv) no sample loss or contamination due to elution or micro-recovery procedures. The model system used is a catalytically active RNA (group IIB intron from Saccharomyces cerevisiae, Sc.ai5{gamma}) and its cofactor Mss116. The protein supports the RNA folding process and thereby the subsequent excision of the intronic RNA from the coding part. Using the novel approach of coupling SPR with MALDI MS, we report the identification of potential RNA-binding proteins from a crude yeast mitochondrial lysate in a non-targeted approach. Our results show that proteins other than the well-known cofactor Mss116 interact with Sc.ai5{gamma} (Dbp8, Prp8, Mrp13, and Cullin-3), suggesting that the intron folding and splicing are regulated by more than one cofactor in vivo. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/082776v3_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@50a09borg.highwire.dtl.DTLVardef@1602253org.highwire.dtl.DTLVardef@6d9cc7org.highwire.dtl.DTLVardef@4ff0f0_HPS_FORMAT_FIGEXP M_FIG C_FIG Author contributionUlrike Anders and Maya Gulotti-Georgieva contributed equally to this work.

molecular biology↗