Liver transplantation recovers hepatic N-glycosylation with persistent IgG glycosylation abnormalities: Three-year follow-up in a patient with phosphomannomutase-2-congenital disorder of glycosylation.

Phosphomannomutase-2-congenital disorder of glycosylation (PMM2-CDG) is the most common CDG and presents with highly variable features ranging from isolated neurologic involvement to severe multi-organ dysfunction. Liver abnormalities occur in in almost all patients and frequently include hepatomegaly and elevated aminotransferases, although only a minority of patients develop progressive hepatic fibrosis and liver failure. No curative therapies are currently available for PMM2-CDG, although investigation into several novel therapies is ongoing. We report the first successful liver transplantation in a 4-year-old patient with PMM2-CDG. Over a 3-year follow-up period, she demonstrated improved growth and neurocognitive development and complete normalization of liver enzymes, coagulation parameters, and carbohydrate-deficient transferrin profile, but persistently abnormal IgG glycosylation and recurrent upper airway infections that did not require hospitalization. Liver transplant should be considered as a treatment option for PMM2-CDG patients with end-stage liver disease, however these patients may be at increased risk for recurrent bacterial infections post-transplant.

The COVID-19 antibody responses, isotypes and glycosylation: Why SARS-CoV-2 Spike protein complex binding of IgG3 is potentiated in some and immuno-pathologies manifest. (preprint)

COVID-19 syndrome does not occur in all who are infected with SARS-CoV-2, and symptoms vary. The anti-SARS CoV-2 Spike immune responses is confounded by the Spike proteins ability to bind Ig{gamma}3 heavy chains. This appears to be via sialic acid glycans found on the O-Linked glycosylation moieties of this heavy chain extended neck domain. Furthermore glycosylation of light chains, particularly Kappa ({kappa}), is an associated feature of antibodies binding to SARS-CoV-2 antigens nucleocapsid and Spike protein. COVID-19 recovered patients had increased IgG1 and IgM levels and un-glycosylated {kappa} {lambda} light chains; possibly In order to counter this immune system subjugation of IgG3. These molecular finding, together with our previous finding that Spike protein binds glycated human serum albumin (HSA), may explain the micro-vascular inflammatory clots that are a causative feature of COVID-19 acute respiratory syndrome (ARDS). The postulated molecular sequelae are that SARS-CoV-2 virion, entering the blood circulation, being coated with IgG3 and glycated HSA forms a colloid and deposits into micro-focal clots which are also inflammatory. It is not that all IgG3 and albumin is being bound by the virus; this depends on the affinity the SARS-CoV2 virion has for binding an individual IgG3 and albumin due to glycosylation and glycation status. The degree of glycosylation and terminal sialyation of an individuals antibodies is both a genetic and age-maturity dependant feature of the immune system. The degree of HSA glycation is also age related feature particularly related to type 2 diabetes. Thereby establishing the molecular basis of the association of severe COVID-19 disease syndrome and deaths with diabetes, metabolic disorders, and old age. Furthermore, already having cardiovascular disease, with hardened arteries, SARS-CoV2-glycated HSA-IgG3 deposition is going to exacerbate an already compromised circulatory physiology. The binding of IgG3 might also drives a shift in the immune repertoire response to SAR-CoV-2 anti-spike antibodies of increased IgG1 and prolonged IgM levels. This may be associated with Long Covid. In summary, SARS-CoV-2 Spike protein binding of IgG3, via sialic acid glycan residues, along with increased glycosylated {kappa}-light chains and glycated-HSA may form a focal amyloid-like precipitate within blood vessels which in turn leads to the inflammatory micro-thrombosis characteristic of COVID-19 immuno-pathology.

Phylodynamic Pattern of Genetic Clusters, Paradigm Shift on Spatio-temporal Distribution of Clades, and Impact of Spike Glycoprotein Mutations of SARS-CoV-2 Isolates from India (preprint)

Background: The COVID-19 pandemic is associated with high morbidity and mortality, with the emergence of numerous variants. The dynamics of SARS-CoV-2 with respect to clade distribution is uneven, unpredictable and fast changing. Aims: Retrieving the complete genomes of SARS-CoV-2 from India and subjecting them to analysis on phylogenetic clade diversity, Spike (S) protein mutations and their functional consequences such as immune escape features and impact on infectivity. Methods: Whole genome of SARS-CoV-2 isolates (n=4,326) deposited from India during the period from January 2020 to December 2020 is retrieved from GISAID and various analyses performed using in silico tools. Results: Notable clade dynamicity is observed indicating the emergence of diverse SARS-CoV-2 variants across the country. GR clade is predominant over the other clades and the distribution pattern of clades is uneven. D614G is the commonest and predominant mutation found among the S-protein followed by L54F. Mutation score prediction analyses reveal that there are several mutations in S-protein including the RBD and NTD regions that can influence the virulence of virus. Besides, mutations having immune escape features as well as impacting the immunogenicity and virulence through changes in the glycosylation patterns are identified. Conclusions: The study has revealed emergence of variants with shifting of clade dynamics within a year in India. It is shown uneven distribution of clades across the nation requiring timely deposition of SARS-CoV-2 sequences. Functional evaluation of mutations in S-protein reveals their significance in virulence, immune escape features and disease severity besides impacting therapeutics and prophylaxis.

Determination of IgG1 and IgG3 SARS-CoV-2 spike protein and nucleocapsid binding – Who is binding who and why? (preprint)

The involvement of IgG3 in the humoral immune response to SARS-CoV2 infection has been implicated in the pathogenesis of ARDS in COVID-19. The exact molecular mechanism is unknown but may be due to the differential ability of IgG3 Fc region to fix complement and stimulate cytokine release. We examined convalescent patients’ antibodies binding to immobilised nucleocapsid and spike protein by MALDI-ToF mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for nucleocapsid versus spike protein demonstrated that the predominant humoral immune response to nucleocapsid was IgG3, whilst against spike it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself which it would bind from control plasma samples as well as from those previously infected with SARS-CoV2, much in the way Protein-G binds IgG1. Furthermore, detailed spectral analysis indicated a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.

Atypical N-glycosylation of SARS-CoV-2 impairs the efficient binding of Spike-RBM to the human-host receptor hACE2 (preprint)

SARS-CoV-2 internalization by human host cells relies on the molecular binding of its spike glycoprotein (SGP) to the angiotensin-converting-enzyme-2 (hACE2) receptor. It remains unknown whether atypical N-glycosylation of SGP modulates SARS-CoV-2 tropism for infections. Here, we address this question through an extensive bioinformatics analysis of publicly available structural and genetic data. We identified two atypical sequons (sequences of N-glycosylation: NGV 481-483 and NGV 501-503), strategically located on the receptor-binding motif (RBM) of SGP and facing the hACE2 receptor. Interestingly, the cryo-electron microscopy structure of trimeric SGP in complex with potent-neutralizing antibodies from convalescent patients revealed covalently-linked N-glycans in NGV 481-483 atypical sequons. Furthermore, NGV 501-503 atypical sequon involves the asparagine-501 residue, whose highly-transmissible mutation N501Y is present in circulating variants of major concerns and affects the SGP-hACE2 binding-interface through the well-known hotspot-353. These findings suggest that atypical SGP post-translational modifications modulate the SGP-hACE2 binding-affinity affecting consequently SARS-CoV-2 transmission and pathogenesis.