BioPharmaSpec Publishes Joint SARS-CoV-2 Research with Imperial College London
Site-specific characterization of SARS-CoV-2 spike glycoprotein receptor binding domain
Malvern, PA, USA and Jersey, GB September 30, 2020 – BioPharmaSpec (“BioPharmaSpec”), a leading contract research organization (CRO) providing a full range of characterization services to the biotechnology industry, today announced it has published a joint paper with Imperial College London on the detailed structural characterization of the SARS CoV-2 Spike Protein Receptor Binding Domain (RBD).
The RBD is an important point of contact between the virus particle and the ACE-2 (Angiotensin Converting Enzyme-2) Receptor on human cells, facilitating the entry of the virus and resulting in infection. The Spike protein sequence has been available for some time and has been used for molecular modelling of this interaction in recent publications, with the aim of assisting understanding of the contact surfaces presumed to be of relevance both to vaccine and drug intervention therapies. However, the recognition that the protein is glycosylated with both N- and O-linked Glycans introduces uncertainty regarding the validity of such purely protein-based modelling data. This is because Glycans, which have a large spatial volume, can conceivably either interfere sterically with potential protein-protein interactions or alternatively enhance the interactions through, for example, hydrogen bonding or charge interaction.
A knowledge of the detailed Glycan structures present on the SARS CoV-2 Spike protein should aid that understanding, and this research just published provides a useful baseline study, beginning with analysis of the “business end” of the molecule produced in a human cell line (HEK293 cells).
Prof. Howard R. Morris, Chairman and CSO of BioPharmaSpec said, “As illustrated in our earlier collaborative viral studies on HIV GP120 Glycosylation, differing constructs may exhibit different Glycosylation patterns, potentially affected by factors specific to cell-type and/or passage-time through the ER and Golgi, and by structure-dependent accessibility to glycosyl transferases and glycosidases. A further source of reported differences, not widely considered at present, may be due to the choice of analytical methods themselves, including mass spectrometer type and mode of operation, or chromatographic separation and overall strategy differences. It will now be important therefore to compare data from differing biological sources and using different analytical methods in order to validate these and other SARS CoV-2 findings.”
In the site-specific work just published, the study used a combined Glycomic and Glycoproteomic strategy refined in the above mentioned HIV work. This involved Q-TOF LC-ESMS and MS/MS analysis of glycoprotein digests for site-specific N- and O- Glycan assignments, together with the production of comparative semi-quantitative N-linked data from MALDI TOF MS and MS/MS analysis of oligosaccharides released from the identified and pooled site-specific LC fractions using PNGaseF, followed by permethylation. In this way, and guided by the MS/MS fragment ions observed, the BioPharmaSpec/ Imperial team identified the presence of unusual epitopes on the Spike protein Receptor Binding Domain in the HEK293 construct, incorporating Fucosylated Lac-DiNAc structures in the antennae at both of the N-linked sites Asn-331 and Asn-343. These were found as significant components amongst an array of mainly Complex-type oligosaccharides, with few if any High-Mannose structures.
Elsewhere in the LC-ESMS and MS/MS data sets, a Di-Sialylated Core-2 structure in high abundance at Thr-323 was we also uniquely identified. This is in contrast to another recent report on HEK293 cell-derived intact Spike protein data showing two Core-1 type substitutions at Thr-323 and Ser-325 respectively. Data generated in this collaborative study on the RBD itself indicated an absence of O-Glycosylation on Ser-325.
Prof. Morris went on to say, “Despite the Spring and early Summer Covid-19 pandemic lock-down restrictions which only allowed only two scientists in a laboratory at any one time, I’m pleased to say that our BioPharmaSpec Laboratories, both in Jersey GB and in Pennsylvania USA, have been able to make a significant contribution in this joint study with Imperial College London, which will be extended in further work to other constructs including the intact Spike protein and its trimer from a range of cell types, and ultimately to materials from viral origin, using this knowledge base as a guide.”
For full explanatory details of this work, please follow the link to the Glycobiology paper together with the Supplementary data, or contact us to find out more.