Abstract 1936: 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.

Polysaccharide degradation by glycoside hydrolases (GHs) contribute to the breakdown of plant biomass needed to support the renewable bioeconomy. Understanding the contributions of structural features and domains allows targets for enzyme enhancement. This work focuses on an enzyme from Acetovibrio thermocellus, CelR, which belongs to glycoside hydrolase family 9 (GH9). Variants of the catalytic domain with native family 3c carbohydrate module (CBM3c), family 3a CBM (CBM3a), both, and neither CBM were expressed and assayed for thermostability, binding, and activity. Thermostability was measured by digital scanning flourimetry protein melting, binding was measured by pull-down of GFP-tagged inactive variants, and activity was measured using a modified BCA assay to detect reducing sugars. Binding to amorphous cellulose in the form of phosphoric acid swollen cellulose (PASC) was strong for all variants and activity on PASC was higher (10x) with CBM3c. Binding to crystalline cellulose (Avicel) was improved greatly (5x) with CBM3a. CBM3a also improved activity (2-5x) on Avicel, however activity was much higher (10x) with CBM3c. CBM3a gave a slight increase in thermostability (+5°C), but CBM3c had a much greater impact on thermostability (+25°C). Continued work is focused on understanding the reaction mechanism of CelR along with another glycoside hydrolase via time resolved serial crystallography. This work reveals the roles of different CBMs and how they interact with CelR. 103750, https://doi.org/10.1016/j.jbc.2023.103750 Abstract 1936 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 Joanne Tobacman, University of Illinois at Chicago

Sumit Bhattacharyya
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.
Results: 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).
Conclusions: 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.

Abstract 1954
The Activity of GFP-Tagged ST6GalNAc-II Against a Series of Differently Charged PSGL-1 Glycopeptides Haley Aharoni, Case Western Reserve University

Collin Ballard, Hana Lee, Thomas Gerken
Mucin-type O-glycosylation is initiated by a family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) that transfers a sugar N-acetylgalactosamine (GalNAc) onto the hydroxyl groups of serine and threonine residue proteins. Studies have shown a correlation between GalNAc-T expression and pathological diseases, like cancer and other diseases. The GalNAc containing Ser and Thr resides can further be elongated by core transferases that add other sugars, while further elongation is terminated through the transfer of a sialic acid onto these glycans via the ST6GalNAc-I&II. Ongoing studies within the Gerken Lab show that flanking charged residues N-and C-terminal to the site of glycosylation may change the glycosylation rates of the GalNAc-Ts and the core elongating transferases. In this study, we used a series of model PSGL-1 glycopeptides to examine the roles of flanking charge residues on the activity of an N-terminal GFP-tagged ST6GalNAc-II transferase. The P-selectin glycoprotein ligand-1 (PSGL-1) is expressed on leukocytes and mediates the rolling of endothelial cells, initiating an inflammatory process. For proper function, PSGL-1 requires a site-specific Oglycan structure, which is a heptasaccharide sidechain containing a Sialyl-Lewis X (SLex) determinant. We observed that the enzyme preferred negatively-charged wild-type glycopeptides over more positive glycopeptides. Our initial hypothesis was that the negative charge of the wild-type PSGL-1 glycopeptide would have reduced ST6GalNAc-II activity, as the early addition of sialic acid on the GalNAc would have terminated SLex glycan elongation. Our findings therefore suggest that other features may play a role in the specificity of GFP-tagged ST6GalNAc-II, which may include the presence of the GFP tag. We acknowledge Kelley Moremen from the University of Georgia for supplying the GFP-tagged ST6GalNAc-II and the NIH Grant R01GM113534 to TAG.