In a recent study published in ACS Central Science, researchers used a luciferase-independent luminescence test to investigate whether the wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein possesses pseudo-luciferase activity for Cypridina luciferin.
Study: Pseudo-Luciferase Activity of the SARS-CoV-2 Spike Protein for Cypridina Luciferin. Image Credit: Myriam B/Shutterstock.com
Background
Luciferin (luminescent substrate) and luciferase (enzyme) are crucial for bioluminescence (BL) detection, allowing for highly selective luminescence detection of target proteins and cells. Imidazopyrazinone-type (IPT) luciferin is found in many marine species, whereas Cypridina luciferase catalyzes Cypridina luciferin and coelenterazine (CTZ). Luciferin emits light in luciferase presence, although it can react with non-luciferase proteins or other biomolecules.
A recent study showed that the CTZ derivative HuLumino can quantitatively detect human serum albumin (HAS) with accuracy equivalent to an enzyme-linked immunosorbent assay (ELISA) in less than a minute.
About the study
In the present study, researchers investigated the application of Cypridina luciferin's oxidative luminescence reaction, catalyzed by the SARS-CoV-2 S protein, in biotechnology. They reported that the SARS-CoV-2 S protein had pseudo-luciferase activity against Cypridina luciferin and investigated its chemical structure and luminescence activity.
The team investigated IPT luciferins for light emission using the full-length monomer of the SARS-CoV-2 S protein. They combined 36 IPT luciferins, including two native luciferins (CTZ and Cypridina luciferin) and 34 previously known CTZ analogs, with S protein. Using the three S protein subunits [S1, S2, and receptor-binding domain (RBD)], the team detected potential Cypridina luciferin components. They compared the kinetic profiles and derived the Km and relative Kcat values from Michaelis-Menten equation fitting curves produced using the starting luminescence intensity for 30 seconds.
The team studied the structure-activity connection of Cypridina luciferin analogs (CLAs) with SARS-CoV-2 spike glycoproteins to gain insights into the luminescence reactions between the Cypridina luciferin substrate and SARS-CoV-2 S glycoproteins. The functional groups, 3-indolyl and 3-(1-guanidino)propyl are unique to Cypridina luciferin and absent in other naturally occurring luciferins. To investigate the effects of the functional chemical groups on SARS-CoV-2 S enzymatic luminescence, the researchers synthesized three CLA types by substituting the NH group of 3-indolyl at C-6 of the ITP ring with a heteroatom and removing 3-(1-guanidino)propyl functional groups from C-8 using synthetic procedures.
The team explored the binding affinity of Cypridina luciferin to the S protein due to the 3-(1-guanidino)propyl functional group at C-8 using computational simulations using Autodock Vina. They also investigated whether a biomolecule-catalyzing chemiluminescence (BCL)-based test method that uses the S protein's pseudo luciferase activity might detect the trimeric SARS-CoV-2 spike glycoprotein in a human salivary sample from a coronavirus disease 2019 (COVID-19) polymerase chain reaction (PCR)-negative donor without sample preprocessing.
Results
The SARS-CoV-2 spike glycoprotein could be identified in human saliva using a BCL-based test method that detects the protein selectively and rapidly without requiring sample preprocessing. Enzymatic identification of the 3-(1-guanidino)propyl functional group in luciferin at the interfaces of the S protein units led to the luminescence response. The BCL approach has the potential to supplement centralized reverse transcription-polymerase chain reaction (RT-PCR) testing, which needs specialized clinical facilities, trained individuals, and lengthy diagnostic timeframes.
The monomeric SARS-CoV-2 S emitted light in Cypridina luciferin presence [signal-to-noise (S/N) ratio of 35) rather than other luciferins. The findings indicated that the SARS-CoV-2 S protein had pseudo-luciferase activity and demonstrated the ability of this technology to supplement centralized testing approaches. The appropriate orthogonal combination of the monomeric SARS-CoV-2 S protein and Cypridina luciferin showed flash-type kinetic reactions, observed in bioluminescent systems utilizing IPT luciferin, with a luminescence intensity drop of roughly 23% over one minute.
IPT luciferin has sec-2-butyl at C-2, 3-indolyl at C-6, and 3-(1-guanidino)propyl functional groups at C-8 sites of the ITP ring, revealing the pseudo luciferase activity of the SARS-CoV-2 spike glycoprotein. Cypridina luciferin, a monomeric spike protein, exhibited a higher efficiency for catalytic reactions than fragment proteins, boosting the relative enzymatic turnover (kcat) values by greater than 2.6. Individual units may not contribute to luciferin luminescence but rather the reaction sites created when units unite. The chemiluminescence system, which produces luciferase-dependent luminescence in aprotic polar fluids, should be classified as BCL based on luminescence intensities.
The Cypridina luciferase (Cluc) and Vargula luciferin combination produced a 30% bioluminescent quantum yield, the highest of any IPT luciferin-based BL system, with reaction specificity. The biomolecule-catalyzing chemiluminescence-based technique identifies SARS-CoV-2 S quantitatively using a "mix-and-read" approach, which involves adding the luciferin protein to the material and monitoring the luminescence signal for one minute. This approach detects the S protein faster and more accurately than the lateral flow assay (LFA) method, which uses S-protein-binding sialic acid.
Conclusion
The study findings revealed a novel method to identify SARS-CoV-2 antigens without genetic alterations or antibodies. Researchers could quantify the pseudo-luciferase activity of SARS-CoV-2 spike glycoproteins in human saliva. The monomeric S protein glows with Cypridina luciferin, but the trimeric S protein needs more luciferin. The 3-indolyl substituent at C-6 and the 3-(1-guanidino)propyl functional group are critical for luminescence activity. The novel protein-analysis technology can detect S proteins in human saliva in one minute without sample preparation.
Genetic risk factors for long-COVID uncovered in a large multi-ethnic study