Increases in expression of carbohydrate sulfotransferases CHST11 and CHST15 and decline in N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB) require phospho-p38-MAPK following exposure to SARS-CoV-2 spike protein receptor binding domain in human a

Objective: Immunohistochemistry of post-mortem lung tissue from Covid-19 patients with diffuse alveolar damage demonstrated marked increases in chondroitin sulfate and CHST15 and decline in N-acetylgalactosamine-4-sulfatase. Studies were undertaken to identify the mechanisms involved in these effects. Method(s): Human primary small airway epithelial cells (PCS 301-010;ATCC) were cultured and exposed to the SARSCoV- 2 spike protein receptor binding domain (SPRBD;AA: Lys310-Leu560;Amsbio). Expression of the spike protein receptor, angiotensin converting enzyme 2 (ACE2), was enhanced by treatment with Interferon-beta. Promoter activation, DNA-binding, RNA silencing, QPCR, Western blots, ELISAs, and specific enzyme inhibitors were used to elucidate the underlying molecular mechanisms. Result(s): Treatment of the cultured cells by the SPRBD led to increased CHST15 and CHST11 expression and decline in ARSB expression. Sulfotransferase activity, total chondroitin sulfate, and sulfated glycosaminoglycan (GAG) content were increased. Phospho-T180/T182-p38-MAPK and phospho- S423/S425-Smad3 were required for the activation of the CHST15 and CHST11 promoters. Inhibition by SB203580, a phospho-p38 MAPK inhibitor, and by SIS3, a Smad3 inhibitor, blocked the CHST15 and CHST11 promoter activation. SB203580 reversed the SPRBD-induced decline in ARSB expression, but SIS3 had no effect on ARSB expression or promoter activation. Phospho-p38 MAPK was shown to reduce retinoblastoma protein (RB) S807/S811 phosphorylation and increase RB S249/T252 phosphorylation. E2F-DNA binding declined following exposure to SPRBD, and SB203580 reversed this effect. This indicates a mechanism by which SPRBD, phospho-p38 MAPK, E2F, and RB can regulate ARSB expression and thereby impact on chondroitin 4-sulfate and dermatan sulfate and molecules that bind to these sulfated GAGs, including Interleukin-8, bone morphogenetic protein-4, galectin-3 and SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2). Conclusion(s): The enzyme ARSB is required for the degradation of chondroitin 4-sulfate and dermatan sulfate, and accumulation of these sulfated GAGs can contribute to lung pathophysiology, as evident in Covid-19. Some effects of the SPRBD may be attributable to unopposed Angiotensin II, when Ang1-7 counter effects are diminished due to binding of ACE2 with the SARS-CoV-2 spike protein and reduced production of Ang1-7. Aberrant cell signaling and activation of the phospho-p38 MAPK and Smad3 pathways increase CHST15 and CHST11 production, which can contribute to increased chondroitin sulfate in infected cells. Decline in ARSB may occur as a consequence of effects of phospho-p38 MAPK on RB phosphorylation and E2F1 availability. Decline in ARSB and the resulting impaired degradation of sulfated GAGs have profound consequences on cellular metabolic, signaling, and transcriptional events. Funding is VA Merit Award.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Profound Impact of Decline in N-Acetylgalactosamine-4-Sulfatase (Arylsulfatase B) on Molecular Pathophysiology and Human Diseases.

The enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) was originally identified as a lysosomal enzyme which was deficient in Mucopolysaccharidosis VI (MPS VI; Maroteaux-Lamy Syndrome). The newly directed attention to the impact of ARSB in human pathobiology indicates a broader, more pervasive effect, encompassing roles as a tumor suppressor, transcriptional mediator, redox switch, and regulator of intracellular and extracellular-cell signaling. By controlling the degradation of chondroitin 4-sulfate and dermatan sulfate by removal or failure to remove the 4-sulfate residue at the non-reducing end of the sulfated glycosaminoglycan chain, ARSB modifies the binding or release of critical molecules into the cell milieu. These molecules, such as galectin-3 and SHP-2, in turn, influence crucial cellular processes and events which determine cell fate. Identification of ARSB at the cell membrane and in the nucleus expands perception of the potential impact of decline in ARSB activity. The regulation of availability of sulfate from chondroitin 4-sulfate and dermatan sulfate may also affect sulfate assimilation and production of vital molecules, including glutathione and cysteine. Increased attention to ARSB in mammalian cells may help to integrate and deepen our understanding of diverse biological phenomenon and to approach human diseases with new insights.

UPDATED RESULTS FROM A NOVEL, LONGITUDINAL, MULTICENTER DISEASE MONITORING PROGRAM (DMP) OF VESTRONIDASE ALFA FOR THE TREATMENT OF MUCOPOLYSACCHARIDOSIS VII (MPS VII)

Background: MPS VII is an ultra-rare, autosomal recessive, debilitating, progressive lysosomal storage disease caused by beta-glucuronidase (GUS) enzyme deficiency. Vestronidase alfa (recombinant human GUS) enzyme replacement therapy is approved in the United States, Europe, and parts of Latin America for the treatment of MPS VII. Methods: The Disease Monitoring Program (DMP) is an ongoing, multicenter observational study collecting standardized real-world data from patients with MPS VII (N ≈ 35 planned) treated with vestronidase alfa or with any other management approach. Investigational sites are centers with expertise in mucopolysaccharidosis treatment. Data will be collected for up to 10 years and include demographics, clinical history, clinical characteristics, cognition, mobility, skeletal disease, pulmonary function, patient and caregiver-reported health-related quality of life, and long-term vestronidase alfa safety and effectiveness. Data are monitored and recorded in compliance with Good Clinical Practice guidelines. Annual individual patient reports will be provided to patients and caregivers. Results: As of 31 May 2021, 20 patients are enrolled: 19 in the treated group and one in the untreated group. Of the 19 treated patients, 14 were treated with vestronidase alfa before DMP enrollment, and five were treated with vestronidase alfa during the DMP. Eleven patients (55%) are male, and 11 patients (55%) are Hispanic or Latino, reflecting that most patients (11 of 20) are enrolled in South America. Mean (SD [range]) age at MPS VII diagnosis was 4.47 (3.99 [0.1-12.0]) years and mean (SD [range]) age at DMP enrollment was 11.08 (7.21 [1.5-26.3]) years. Seven patients (35%) had a history of non-immune hydrops fetalis. For the 14 patients treated prior to DMP enrollment, mean (SD [range]) age at initiation of vestronidase alfa was 9.4 (6.3 [3.0-22.8]) years. For the five patients treated during the DMP, mean (SD [range]) age at initiation of vestronidase alfa was 6.1 (4.5 [2.5-11.5]) years. Three patients who reached two years of treatment in the DMP had an 88% and 75% reduction from baseline in the original (parent) clinical study in dermatan sulfate and chondroitin sulfate uGAG excretion, respectively. Four serious adverse events (SAEs) in two patients have been reported. One SAE, intermittent hypotension, was assessed as an infusion-associated reaction to vestronidase alfa;this SAE did not meet hypersensitivity criteria;this patient had a low positive anti-drug antibody (ADA) titer (1:80) prior to the first administration of vestronidase alfa but did not test ADA positive at any subsequent visit during the DMP. All SAEs were consistent with the known safety profile of vestronidase alfa. No deaths were reported. COVID-19 resulted in travel restrictions for many patients, but only one dose of vestronidase alfa was missed. Nine patients had ADA data analyzed at baseline;of these, four tested positive at initial baseline, but all four subsequently tested negative during the DMP, while three other patients had low positive ADA titers (range: 1:10 to 1:320). All patients with positive ADA samples tested negative for neutralizing ADA, and four had reductions of at least 80% in dermatan sulfate uGAG excretion from baseline in the original (parent) clinical study. Conclusions: Reductions in uGAG demonstrate ongoing effectiveness of vestronidase alfa at DMP Year 2. No new safety concerns were identified, and all patients continue on study. The MPS VII DMP continues to enroll patients and collect data to characterize MPS VII disease presentation, clinical heterogeneity, and progression.

An Autoantigen Atlas From Human Lung HFL1 Cells Offers Clues to Neurological and Diverse Autoimmune Manifestations of COVID-19.

COVID-19 is accompanied by a myriad of both transient and long-lasting autoimmune responses. Dermatan sulfate (DS), a glycosaminoglycan crucial for wound healing, has unique affinity for autoantigens (autoAgs) from apoptotic cells. DS-autoAg complexes are capable of stimulating autoreactive B cells and autoantibody production. We used DS-affinity proteomics to define the autoantigen-ome of lung fibroblasts and bioinformatics analyses to study the relationship between autoantigenic proteins and COVID-induced alterations. Using DS-affinity, we identified an autoantigen-ome of 408 proteins from human HFL1 cells, at least 231 of which are known autoAgs. Comparing with available COVID data, 352 proteins of the autoantigen-ome have thus far been found to be altered at protein or RNA levels in SARS-CoV-2 infection, 210 of which are known autoAgs. The COVID-altered proteins are significantly associated with RNA metabolism, translation, vesicles and vesicle transport, cell death, supramolecular fibrils, cytoskeleton, extracellular matrix, and interleukin signaling. They offer clues to neurological problems, fibrosis, smooth muscle dysfunction, and thrombosis. In particular, 150 altered proteins are related to the nervous system, including axon, myelin sheath, neuron projection, neuronal cell body, and olfactory bulb. An association with the melanosome is also identified. The findings from our study illustrate a connection between COVID infection and autoimmunity. The vast number of COVID-altered proteins with high intrinsic propensity to become autoAgs offers an explanation for the diverse autoimmune complications in COVID patients. The variety of autoAgs related to mRNA metabolism, translation, and vesicles suggests a need for long-term monitoring of autoimmunity in COVID. The COVID autoantigen atlas we are establishing provides a detailed molecular map for further investigation of autoimmune sequelae of the pandemic, such as "long COVID" syndrome. Summary Sentence: An autoantigen-ome by dermatan sulfate affinity from human lung HFL1 cells may explain neurological and autoimmune manifestations of COVID-19.

A master autoantigen-ome links alternative splicing, female predilection, and COVID-19 to autoimmune diseases.

Chronic and debilitating autoimmune sequelae pose a grave concern for the post-COVID-19 pandemic era. Based on our discovery that the glycosaminoglycan dermatan sulfate (DS) displays peculiar affinity to apoptotic cells and autoantigens (autoAgs) and that DS-autoAg complexes cooperatively stimulate autoreactive B1 cell responses, we compiled a database of 751 candidate autoAgs from six human cell types. At least 657 of these have been found to be affected by SARS-CoV-2 infection based on currently available multi-omic COVID data, and at least 400 are confirmed targets of autoantibodies in a wide array of autoimmune diseases and cancer. The autoantigen-ome is significantly associated with various processes in viral infections, such as translation, protein processing, and vesicle transport. Interestingly, the coding genes of autoAgs predominantly contain multiple exons with many possible alternative splicing variants, short transcripts, and short UTR lengths. These observations and the finding that numerous autoAgs involved in RNA-splicing showed altered expression in viral infections suggest that viruses exploit alternative splicing to reprogram host cell machinery to ensure viral replication and survival. While each cell type gives rise to a unique pool of autoAgs, 39 common autoAgs associated with cell stress and apoptosis were identified from all six cell types, with several being known markers of systemic autoimmune diseases. In particular, the common autoAg UBA1 that catalyzes the first step in ubiquitination is encoded by an X-chromosome escape gene. Given its essential function in apoptotic cell clearance and that X-inactivation escape tends to increase with aging, UBA1 dysfunction can therefore predispose aging women to autoimmune disorders. In summary, we propose a model of how viral infections lead to extensive molecular alterations and host cell death, autoimmune responses facilitated by autoAg-DS complexes, and ultimately autoimmune diseases. Overall, this master autoantigen-ome provides a molecular guide for investigating the myriad of autoimmune sequalae to COVID-19 and clues to the rare adverse effects of the currently available mRNA and viral vector-based COVID vaccines.

Enoxaparin and Pentosan Polysulfate Bind to the SARS-CoV-2 Spike Protein and Human ACE2 Receptor, Inhibiting Vero Cell Infection.

As with many other pathogens, SARS-CoV-2 cell infection is strongly dependent on the interaction of the virus-surface Spike protein with the glycosaminoglycans of target cells. The SARS-CoV-2 Spike glycoprotein was previously shown to interact with cell-surface-exposed heparan sulfate and heparin in vitro. With the aim of using Enoxaparin as a treatment for COVID-19 patients and as prophylaxis to prevent interpersonal viral transmission, we investigated GAG binding to the Spike full-length protein, as well as to its receptor binding domain (RBD) in solution by isothermal fluorescence titration. We found that Enoxaparin bound to both protein variants with similar affinities, compared to the natural GAG ligand heparan sulfate (with Kd-values in the range of 600-680 nM). Using size-defined Enoxaparin fragments, we discovered the optimum binding for dp6 or dp8 for the full-length Spike protein, whereas the RBD did not exhibit a significant chain-length-dependent affinity for heparin oligosaccharides. The soluble ACE2 receptor was found to interact with unfractionated GAGs in the low µM Kd range, but with size-defined heparins with clearly sub-µM Kd-values. Interestingly, the structural heparin analogue, pentosan polysulfate (PPS), exhibited high binding affinities to both Spike variants as well as to the ACE2 receptor. In viral infection experiments, Enoxaparin and PPS both showed a strong inhibition of infection in a concentration range of 50-500 µg/mL. Both compounds were found to retain their inhibitory effects at 500 µg/mL in a natural biomatrix-like human sputum. Our data suggest the early topical treatment of SARS-CoV-2 infections with inhaled Enoxaparin; some clinical studies in this direction are already ongoing, and they further imply an oral or nasal prophylactic inactivation of the virus by Enoxaparin or PPS for the prevention of inter-personal viral transmission.

Home treatment of type VI mucopolysaccharidosis (Maroteaux-Lamy syndrome) an alternative at this time of COVID-19 pandemic: A case in Peru.

We report a patient with mucopolysaccharidosis type VI, on long-term enzyme replacement home therapy. Results support the efficacy and safety benefits, with additional advantage of home therapy to minimize the risk of community-transmitted infections.

Comparative Anticoagulant and Thrombin Generation Inhibitory Profile of Heparin, Sulodexide and Its Components.

INTRODUCTION: Sulodexide represents a mixture of fast-moving heparin (FMH) and dermatan sulfate (DS) and has been used for the management of venous diseases such as DVT and related disorders. The purpose of this study is to compare sulodexide and its components with unfractionated heparin (UFH) to determine its suitability for the indications in which UFH is used. MATERIALS AND METHOD: Active pharmaceutical ingredients (API) versions of sulodexide, FMH and DS were obtained from Alfasigma. API versions of UFH were obtained from Medefil Inc. Normal human citrated plasma was obtained from blood bank of the Loyola University Medical Center. Each of the individual agents were supplemented in plasma at a graded concentration of 0.0-10 µg/mL. Clotting assays (PiCT, aPTT, PT and TT), anti-Xa and anti-IIa and thrombin generation studies were carried out. Results were compiled as mean ± SD of 3 individual determination. RESULT: In the clot based (PiCT, aPTT and TT), anti-Xa and IIa assays, both the UFH and FMH produced stronger activities in these assays followed by sulodexide. DS did not show any anticoagulant activity. In the thrombin generation assay, FMH and UFH produced comparable inhibition of thrombin generation as measured by various parameters. Sulodexide was slightly weaker in this assay, whereas DS produced relatively weaker effects. CONCLUSION: In comparison to sulodexide, both UFH and FMH exhibit comparable anticoagulant activity despite differences in their molecular weight. These results suggest that sulodexide can be developed as a parenteral anticoagulant for indications in which UFH is used.