Work

N-Glycosylation in the Receptor Binding Domain serves as aggregation gatekeeper of the SARS CoV2 Spike glycoprotein folding

Year: 2026

Type: article

Abstract: The SARS CoV2 Spike is a multi-domain surface glycoprotein that binds human ACE2 via its receptor binding domain (RBD-CoV2) initiating the viral infection. Several aggregation prone regions are predicted in the Spike-CoV2 sequence. Here, we describe the intrinsic aggregation feature and characterization of RBD-CoV2 aggregates of the Wuhan variant in the context of its folding. In agreement with its predicted aggregation-prone nature, recombinant RBD-CoV2 in E. coli exclusively forms inclusion bodies that binds Thioflavin S dye. In vitro refolding of pure denatured RBD-CoV2 results in extensive aggregation yielding very low amounts of soluble functional protein. The RBD-CoV2 in vitro aggregates contain β-sheet structure as revealed by the Raman spectral analysis, bind amyloid-specific AmyTracker 630 and ProteoStat dyes, and have clustered sphere-like morphology. Similarly, the non-glycosylated RBD-CoV2 missing the N-glycan on Asn331 and Asn343, an equivalent to EcRBD, is not secreted, but retained in the endoplasmic reticulum of HEK293T cells. The ProteoStat dye binding indicates intracellular accumulation of the N-glycan RBD variants as amyloids in live cells. Furthermore, selective removal of both RBD N-glycans results in severe secretion defect of the Spike ectodomain, and markedly reduced syncytia formation in cells expressing glycan variants of membrane-bound Spike. Our study thus conclusively demonstrates that the essential role of RBD N-glycans is to prevent misfolding and aggregation of RBD-CoV2 and Spike-CoV2. The presence of N-glycan at either one of the two Asn in RBD significantly reduces misfolding of the full length Spike-CoV2, and the absence of both glycans results in RBD-CoV2 to form amyloids.

Source: Journal of Biological Chemistry

DOI: 10.1016/j.jbc.2026.113681

Authors: Anita Kumari, Beena Krishnan

Institutions: Institute of Microbial Technology, Council of Scientific and Industrial Research, Academy of Scientific and Innovative Research

Language: English

Topic: SARS-CoV-2 and COVID-19 Research

Subfield: Infectious Diseases

Field: Medicine

Domain: Health Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $3,430