bims-supasi 2023-12-10 papers

Anim Sci J. 2023 Jan-Dec;94(1):94(1): e13894

Qualitative and quantitative analyses in sulfated glycosaminoglycans, chondroitin sulfate/dermatan sulfate, during 3 T3-L1 adipocytes differentiation.

Danang Dwi Cahyadi, Katsuhiko Warita, Naoko Takeda-Okuda, Jun-Ichi Tamura, Yoshinao Z Hosaka.

Chondroitin sulfate/dermatan sulfate (CS/DS) is a member of glycosaminoglycans (GAGs) found in animal tissues. Major CS/DS subclasses, O, A, C, D, and E units, exist based on the sulfation pattern in d-glucuronic acid (GlcA) and N-acetyl-d-galactosamine repeating units. DS is formed when GlcA is epimerized into l-iduronic acid. Our study aimed to analyze the CS/DS profile in 3 T3-L1 cells before and after adipogenic induction. CS/DS contents, molecular weight (Mw), and sulfation pattern were analyzed by using high-performance liquid chromatography. CS/DS synthesis- and sulfotransferase-related genes were analyzed by reverse transcription real-time PCR. CS/DS amount was significantly decreased in the differentiated (DI) group compared to the non-differentiated (ND) group, along with a lower expression of CS biosynthesis-related genes, chondroitin sulfate N-acetylgalactosaminyltransferase 1 and 2, as well as chondroitin polymerizing factor. GAGs in the DI group also showed lower Mw than those of ND. Furthermore, the A unit was the major CS/DS in both groups, with a proportionally higher CS-A in the DI group. This was consistent with the expression of carbohydrate sulfotransferase 12 that encodes chondroitin 4-O-sulfotransferase, for CS-A formation. These qualitative and quantitative changes in CS/DS and CS/DS-synthases before and after adipocyte differentiation reveal valuable insights into adipocyte development.

Keywords: 3 T3-L1; adipocytes; adipogenesis; chondroitin sulfate; glycosaminoglycans

DOI: https://doi.org/10.1111/asj.13894

bioRxiv. 2023 Nov 22. pii: 2023.11.22.568358. [Epub ahead of print]

Human extracellular sulfatases use a dual mechanism for regulation of growth factor interactions with heparan sulfate proteoglycans.

Bryce M Timm, Julianna L Follmar, Ryan N Porell, Kimberly Glass, Bryan E Thacker, Charles A Glass, Kamil Godula.

Membrane-associated heparan sulfate (HS) proteoglycans (PGs) contribute to the regulation of extracellular cellular signaling cues, such as growth factors (GFs) and chemokines, essential for normal organismal functions and implicated in various pathophysiologies. PGs accomplish this by presenting high affinity binding sites for GFs and their receptors through highly sulfated regions of their HS polysaccharide chains. The composition of HS, and thus GF-binding specificity, are determined during biosynthetic assembly prior to installation at the cell surface. Two extracellular 6- O -endosulfatase enzymes (Sulf-1 and Sulf-2) can uniquely further edit mature HS and alter its interactions with GFs by removing specific sulfation motifs from their recognition sequence on HS. Despite being implicated as signaling regulators during development and in disease, the Sulfs have resisted structural characterization, and their substrate specificity and effects on GF interactions with HS are still poorly defined. Using a panel of PG-mimetics comprising compositionally-defined bioengineered recombinant HS (rHS) substrates in combination with GF binding and enzyme activity assays, we have discovered that Sulfs control GF-HS interactions through a combination of catalytic processing and competitive blocking of high affinity GF-binding sites, providing a new conceptual framework for understanding the functional impact of these enzymes in biological context. Although the contributions from each mechanism are both Sulf- and GF-dependent, the PG-mimetic platform allows for rapid analysis of these complex relationships.
Significance Statement: Cells rely on extracellular signals such as growth factors (GFs) to mediate critical biological functions. Membrane-associated proteins bearing negatively charged heparan sulfate (HS) sugar chains engage with GFs and present them to their receptors, which regulates their activity. Two extracellular sulfatase (Sulf) enzymes can edit HS and alter GF interactions and activity, although the precise mechanisms remain unclear. By using chemically defined HS-mimetics as probes, we have discovered that Sulfs can modulate HS by means of catalytic alterations and competitive blocking of GF-binding sites. These unique dual activities distinguish Sulfs from other enzymes and provide clues to their roles in development and disease.

DOI: https://doi.org/10.1101/2023.11.22.568358

Genet Mol Biol. 2023 ;pii: S1415-47572023000600109. [Epub ahead of print]46(3 Suppl 1): e20230117

In vitro substrate reduction, chaperone and immunomodulation treatments reduce heparan sulfate in mucolipidosis III human fibroblasts.

Fernanda Sperb-Ludwig, Nataniel Floriano Ludwig, Gustavo Mottin Rizowy, Renata Voltolini Velho, Ida Vanessa Doederlein Schwartz.

Mucolipidosis II and III (MLII and MLIII) are autosomal recessive diseases caused by pathogenic variants in GNPTAB and GNPTG genes that lead to defects in GlcNAc-1-phosphotransferase. This enzyme adds mannose 6-phosphate residues to lysosomal hydrolases, which allows enzymes to enter lysosomes. Defective GlcNAc-1-phosphotransferase causes substrate accumulation and inflammation. These diseases have no treatment, and we hypothesized that the use of substrate reduction therapy and immunomodulation may be beneficial at the cell level and as a future therapeutic approach. Fibroblasts from two patients with MLIII alpha/beta and 2 patients with MLIII gamma as well as from one healthy control were treated with 10 µM miglustat, 20 µM genistein, and 20 µM thalidomide independently. ELISA assay and confocal immunofluorescence microscopy were used to evaluate the presence of heparan sulfate (HS) and the impact on substrate accumulation. ELISA assay showed HS reduction in all patients with the different treatments used (p=0.05). HS reduction was also observed by immunofluorescence microscopy. Our study produced encouraging results, since the reduction in substrate accumulation, even partial, may offer benefits to the phenotype of patients with inborn errors of metabolism.

DOI: https://doi.org/10.1590/1678-4685-GMB-2023-0117

Nano Lett. 2023 Dec 06.

Heparin-Induced Allosteric Changes in SARS-CoV-2 Spike Protein Facilitate ACE2 Binding and Viral Entry.

Simon J L Petitjean, Savannah Eeckhout, Martin Delguste, Qingrong Zhang, Kimberley Durlet, David Alsteens.

Understanding the entry of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) into host cells is crucial in the battle against COVID-19. Using atomic force microscopy (AFM), we probed the interaction between the virus's spike protein and heparan sulfate (HS) as a potential attachment factor. Our AFM studies revealed a moderate-affinity interaction between the spike protein and HS on both model surfaces and living cells, highlighting HS's role in early viral attachment. Remarkably, we observed an interplay between HS and the host cell receptor angiotensin-converting enzyme 2 (ACE2), with HS engagement resulting in enhanced ACE2 binding and subsequent viral entry. Our research furthers our understanding of SARS-CoV-2 infection mechanisms and reveals potential interventions targeting viral entry. These insights are valuable as we navigate the evolving landscape of viral threats and seek effective strategies to combat emerging infectious diseases.

Keywords: ACE2; SARS-CoV-2; atomic force microscopy; heparin; viral entry

DOI: https://doi.org/10.1021/acs.nanolett.3c03550

Discov Med. 2023 Dec;35(179): 1147-1159

Downregulation of HS6ST2 Inhibits Cervical Cancer Cell Migration and Invasion in Vivo and in Vitro.

Shanfeng Li, Haichuan Shen, Qingzhi Wan, Pinjing Chen, Chenggan Shu, Hongmei Wang, Weipei Zhu.

BACKGROUND: Emerging evidence indicates the importance of heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) in a number of developmental processes. Little is known regarding its biological function in regulating cervical cancer (CC) progression. In this study, we aim to explore the role of HS6ST2 in CC progression.
METHODS: The transcriptome sequencing data of CC tissues from three databases, GSE64217, GSE138080, and GSE63514, was examined for genes with significant changes. The expression profile for HS6ST2 within CC tissue was then assessed through fluorescence quantitative PCR and immunohistochemistry and compared to data from patients with clinicopathological features. A multivariate survival analysis was performed using the COX regression. The real-time quantitative PCR assessed the HS6ST2 expression profile within CC cellular cultures. The results of knocking down HS6ST2, considering the proliferative activity and invasiveness of CC cultures in vitro, were detected through cell viability assay, clonogenic assessment, tumorsphere formation analysis, 3D invasion experiment and transwell assay. The impact of HS6ST2 knockdown in CC proliferation was also evaluated in vivo using a nude mice model.
RESULTS: HS6ST2 was severely upregulated within CC tissues across the three explored databases (GSE64217, GSE138080, and GSE63514). Fluorescent quantitative PCR and immunohistochemistry experiments identified HS6ST2 as highly upregulated within patients CC tissues. Survival analysis taking into account the parameters of lymph node metastasis, Federation of Gynecology and Obstetrics (FIGO) stage, depth of invasion, pathological grade, and HS6ST2 expression level demonstrated that individuals with downregulated HS6ST2 exhibited considerably extended progression-free survival (PFS) and overall survival (OS) in comparison to upregulated HS6ST2 cases. According to the findings of COX univariate analysis, the parameters lymph node metastasis, FIGO stage, depth of invasion, pathological grade, and HS6ST2 expression level, all showed a statistically significant correlation with effect upon prognosis of CC patients. The FIGO stage, depth of invasion and expression level of HS6ST2 were identified as independent risk variables influencing CC case prognosis within subsequent COX multivariate analysis. Cell function experiments proved that HS6ST2 knockdown can considerably diminish the proliferative potential, stemness and invasive traits of CC cells. Tumor formation experiments in nude mice in vivo demonstrated that knocking down HS6ST2 can significantly thwart CC cellular proliferative properties within animal models.
CONCLUSIONS: The clinicopathological features and the survival time of the patients significantly correlate with the level of HS6ST2 expression in CC tissue samples.

Keywords: HS6ST2; cervical cancer; invasion; proliferation

DOI: https://doi.org/10.24976/Discov.Med.202335179.111

Chembiochem. 2023 Dec 06. e202300744

Effect of Sulfotyrosine and Negatively Charged Amino Acid of Leech-Derived Peptides on Binding and Inhibitory Activity Against Thrombin.

Tzu-Yin Chen, Eileen Shyur, Tzu-Hsuan Ma, Lakshmi Wijeyewickrema, Sheng-Wei Lin, Mu-Rong Kao, Pi-Hui Liang, Jiun-Jie Shie, Er-Yuan Chuang, Jing-Ping Liou, Yves Sy Hsieh.

Hirudins, natural sulfo(glyco)proteins, are clinical anticoagulants that directly inhibit thrombin, a key coagulation factor. Their potent thrombin inhibition primarily results from antagonistic interactions with both the catalytic and non-catalytic sites of thrombin. Hirudins often feature sulfate moieties on Tyr residues in their anionic C-terminus region, enabling strong interactions with thrombin exosite-I and effectively blocking its engagement with fibrinogen. Although sulfotyrosines have been identified in various hirudin variants, the precise relationship between sulfotyrosine and the number of negatively charged amino acids within the anionic-rich C-terminus peptide domain for the binding of thrombin has remained elusive. By using Fmoc-SPPS, hirudin dodecapeptides homologous to the C-terminus of hirudin variants from various leech species were successfully synthesized, and the effect of sulfotyrosine and the number of negatively charged amino acids on hirudin-thrombin interactions was investigated. Our findings did not reveal any synergistic effect between an increasing number of sulfotyrosines or negatively charged amino acids and their inhibitory activity on thrombin or fibrinolysis in the assays, despite a higher binding level toward thrombin in the sulfated dodecapeptide Hnip_Hirudin was observed in SPR analysis.

Keywords: Hirudin * Sulfotyrosine * negatively charged amino acid * thrombin

DOI: https://doi.org/10.1002/cbic.202300744

Nat Commun. 2023 Dec 06. 14(1): 8069

Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function.

Horacio Maldonado, Bryan D Savage, Harlan R Barker, Ulrike May, Maria Vähätupa, Rahul K Badiani, Katarzyna I Wolanska, Craig M L Turner, Toini Pemmari, Tuomo Ketomäki, Stuart Prince, Martin J Humphries, Erkki Ruoslahti, Mark R Morgan, Tero A H Järvinen.

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro. The heparan sulfate proteoglycan syndecan-4 regulates cell migration and is crucial for wound healing. We report that syndecan-4 expression is restricted to epidermis and blood vessels in mice skin wounds. Syndecan-4 regulates binding and internalisation of CAR peptide and CAR-mediated cytoskeletal remodelling. CAR induces syndecan-4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor cytohesin-2, and promotes syndecan-4-, ARF6- and Cytohesin-2-mediated keratinocyte migration. Finally, we show that genetic ablation of syndecan-4 in male mice eliminates CAR-induced wound re-epithelialisation following systemic administration. We propose that CAR peptide activates syndecan-4 functions to selectively promote re-epithelialisation. Thus, CAR peptide provides a therapeutic approach to enhance wound healing in mice; systemic, yet target organ- and cell-specific.

DOI: https://doi.org/10.1038/s41467-023-43848-1