Martyna Maszota‐Zieleniak, Adam Liwo, Sylvie Ricard‐Blum, Sergey A. Samsonov
Abstract We have previously shown that the extracellular domains of the four syndecans are intrinsically disordered, and adopt a wide range of conformations. We report here the building of coarse‐grained models of the extracellular domains of human syndecans 2 and 4 using small‐angle X‐ray scattering restraints. One, two or three heparan sulfate (HS) hexadecasaccharides, (IdoA[2S]GlcNS[6S]) 8 , were attached to three serine residues of the core proteins, resulting in eight variants for each syndecan that were used for all‐atom molecular dynamics (MD) simulations (0.5–1 µs). Syndecan‐4 had a larger conformational diversity than syndecan‐2, and remained extended during MD simulations in absence of HS whereas syndecan‐2 adopted more compact conformations. Their core proteins thus appeared to be structurally distinct. The HS chains also behave differently, the middle chain being more flexible in syndecan‐4, and the third chain being able to interact with the core protein regions mediating cell adhesion. The cell adhesion sites on both core proteins were flexible, with or without HS chains, the NXIP motif of syndecan‐2 being located in a particularly flexible region. In conclusion, the HS chains induce moderate changes in the conformational dynamics of both syndecans, depending on the number of HS chains and their location on the core protein, and on the core protein itself.
{"title":"Interplay of heparan sulfate chains with the core proteins of syndecans 2 and 4","authors":"Martyna Maszota‐Zieleniak, Adam Liwo, Sylvie Ricard‐Blum, Sergey A. Samsonov","doi":"10.1002/pgr2.10","DOIUrl":"https://doi.org/10.1002/pgr2.10","url":null,"abstract":"Abstract We have previously shown that the extracellular domains of the four syndecans are intrinsically disordered, and adopt a wide range of conformations. We report here the building of coarse‐grained models of the extracellular domains of human syndecans 2 and 4 using small‐angle X‐ray scattering restraints. One, two or three heparan sulfate (HS) hexadecasaccharides, (IdoA[2S]GlcNS[6S]) 8 , were attached to three serine residues of the core proteins, resulting in eight variants for each syndecan that were used for all‐atom molecular dynamics (MD) simulations (0.5–1 µs). Syndecan‐4 had a larger conformational diversity than syndecan‐2, and remained extended during MD simulations in absence of HS whereas syndecan‐2 adopted more compact conformations. Their core proteins thus appeared to be structurally distinct. The HS chains also behave differently, the middle chain being more flexible in syndecan‐4, and the third chain being able to interact with the core protein regions mediating cell adhesion. The cell adhesion sites on both core proteins were flexible, with or without HS chains, the NXIP motif of syndecan‐2 being located in a particularly flexible region. In conclusion, the HS chains induce moderate changes in the conformational dynamics of both syndecans, depending on the number of HS chains and their location on the core protein, and on the core protein itself.","PeriodicalId":74585,"journal":{"name":"Proteoglycan research","volume":"728 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136260478","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Correction to “Functional and structural insights into human <i>N</i>‐deacetylase/<i>N</i>‐sulfotransferase activities”","authors":"","doi":"10.1002/pgr2.12","DOIUrl":"https://doi.org/10.1002/pgr2.12","url":null,"abstract":"","PeriodicalId":74585,"journal":{"name":"Proteoglycan research","volume":"97 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135857748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"The role of hyperglycemia‐evoked intracellular hyaluronan accumulation and its activity on the autophagic and endoplasmic reticulum stress pathways","authors":"A. Wang, Aimin Wang, V. Hascall","doi":"10.1002/pgr2.7","DOIUrl":"https://doi.org/10.1002/pgr2.7","url":null,"abstract":"","PeriodicalId":74585,"journal":{"name":"Proteoglycan research","volume":"88 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84421742","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sylvain D. Vallet, T. Annaval, R. Vivès, Emeline Richard, Jérôme Hénault, C. Le Narvor, D. Bonnaffé, B. Priem, R. Wild, H. Lortat‐Jacob
Heparan sulfate (HS) is a linear polysaccharide composed of a glucuronic acid (GlcA)‐N‐acetyl‐glucosamine (GlcNAc) disaccharide repeat motif, polymerized by the EXT1–EXT2 complex. It is extensively modified by a series of Golgi localized enzymes, that generate distinct saccharide sequences involved in the binding and the regulation of numerous protein partners. N‐deacetylase/N‐sulfotransferase (NDST), of which four isoforms have been identified in mammals, are involved in the first step of this process and catalyze both the N‐deacetylation of the GlcNAc residues into GlcNH2 and its re‐N‐sulfation into GlcNS residues. Further modifications of the HS chain depend on this first maturation event, NDST action is, therefore, key to HS biosynthesis. However, although the sulfotransferase domain of NDST1 has been characterized at the structural level some 20 years ago, information on the overall structure and activity of the enzyme are still lacking. Here, we report the characterization of the two most expressed NDSTs in humans, NDST1 and NDST2, and a model structure of NDST1 homodimer using cryoelectron microscopy combined with AlphaFold2 modeling. Structure‐driven mutagenesis along with two bioassays to follow the protein activities allowed us to characterize the kinetics of the deacetylation and sulfoaddition and to identify the residue H529 as necessary for N‐deacetylation. These results shed light on a poorly understood family of enzymes and will help deciphering the molecular basis for HS and heparin maturation.
硫酸肝素(HS)是由葡萄糖醛酸(GlcA) - N -乙酰-氨基葡萄糖(GlcNAc)双糖重复基序组成的线性多糖，由EXT1-EXT2络合物聚合而成。它被一系列高尔基定位酶广泛修饰，这些酶产生不同的糖序列，参与许多蛋白质伴侣的结合和调节。N -去乙酰化酶/N -磺基转移酶(NDST)参与了这一过程的第一步，并催化GlcNAc残基的N -去乙酰化转化为GlcNH2和其再- N -磺化转化为GlcNS残基。NDST在哺乳动物中已鉴定出四种亚型。HS链的进一步修饰依赖于这第一个成熟事件，因此NDST作用是HS生物合成的关键。然而，尽管NDST1的硫转移酶结构域在20多年前就已经在结构水平上得到了表征，但关于该酶的整体结构和活性的信息仍然缺乏。在这里，我们报道了两种在人类中表达最多的NDST1和NDST2的特征，以及NDST1同型二聚体的模型结构，使用冷冻电镜结合AlphaFold2模型。结构驱动诱变以及跟踪蛋白质活性的两次生物测定使我们能够表征脱乙酰化和磺化的动力学，并确定残留物H529是N -去乙酰化所必需的。这些结果揭示了一个鲜为人知的酶家族，并将有助于破译HS和肝素成熟的分子基础。
{"title":"Functional and structural insights into human N‐deacetylase/N‐sulfotransferase activities","authors":"Sylvain D. Vallet, T. Annaval, R. Vivès, Emeline Richard, Jérôme Hénault, C. Le Narvor, D. Bonnaffé, B. Priem, R. Wild, H. Lortat‐Jacob","doi":"10.1002/pgr2.8","DOIUrl":"https://doi.org/10.1002/pgr2.8","url":null,"abstract":"Heparan sulfate (HS) is a linear polysaccharide composed of a glucuronic acid (GlcA)‐N‐acetyl‐glucosamine (GlcNAc) disaccharide repeat motif, polymerized by the EXT1–EXT2 complex. It is extensively modified by a series of Golgi localized enzymes, that generate distinct saccharide sequences involved in the binding and the regulation of numerous protein partners. N‐deacetylase/N‐sulfotransferase (NDST), of which four isoforms have been identified in mammals, are involved in the first step of this process and catalyze both the N‐deacetylation of the GlcNAc residues into GlcNH2 and its re‐N‐sulfation into GlcNS residues. Further modifications of the HS chain depend on this first maturation event, NDST action is, therefore, key to HS biosynthesis. However, although the sulfotransferase domain of NDST1 has been characterized at the structural level some 20 years ago, information on the overall structure and activity of the enzyme are still lacking. Here, we report the characterization of the two most expressed NDSTs in humans, NDST1 and NDST2, and a model structure of NDST1 homodimer using cryoelectron microscopy combined with AlphaFold2 modeling. Structure‐driven mutagenesis along with two bioassays to follow the protein activities allowed us to characterize the kinetics of the deacetylation and sulfoaddition and to identify the residue H529 as necessary for N‐deacetylation. These results shed light on a poorly understood family of enzymes and will help deciphering the molecular basis for HS and heparin maturation.","PeriodicalId":74585,"journal":{"name":"Proteoglycan research","volume":"26 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87913298","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Functional organization of extracellular hyaluronan, CD44, and RHAMM","authors":"M. Cowman, E. Turley","doi":"10.1002/pgr2.4","DOIUrl":"https://doi.org/10.1002/pgr2.4","url":null,"abstract":"","PeriodicalId":74585,"journal":{"name":"Proteoglycan research","volume":"171 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76612066","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-01-01Epub Date: 2023-03-28DOI: 10.1002/pgr2.1
Kaushlendra Tripathi, Shyam K Bandari, Ralph D Sanderson
Heparanase is upregulated during the progression of most cancers and via its enzyme activity promotes extracellular matrix degradation, angiogenesis and cell migration. Heparanase expression is often associated with enhanced tumor aggressiveness and chemoresistance. We previously demonstrated that increased heparanase expression in tumor cells enhances secretion and alters the composition of tumor-released exosomes. In the present study, we discovered that extracellular vesicles (EVs) secreted by human multiple myeloma cells growing in hypoxic conditions exhibited elevated levels of heparanase cargo compared to EVs from cells growing in normoxic conditions. When macrophages (RAW 264.7 monocyte/macrophage-like cells) were exposed to EVs released by tumor cells growing in either hypoxic or normoxic conditions, macrophage migration and invasion was elevated by EVs from hypoxic conditions. The elevated invasion of macrophages was blocked by a monoclonal antibody that inhibits heparanase enzyme activity. Moreover, the heparanase-bearing EVs from hypoxic cells greatly enhanced endothelial cell tube formation consistent with the known role of heparanase in promoting angiogenesis. EVs from hypoxic tumor cells when compared with EVs from normoxic cells also enhanced cancer stemness properties of both CAG and RPMI 8226 human myeloma cells. Together these data indicate that under hypoxic conditions, tumor cells secrete EVs having an elevated level of heparanase as cargo. These EVs can act on both tumor and non-tumor cells, enhancing tumor progression and tumor cell stemness that likely supports chemoresistance and relapse of tumor.
{"title":"Extracellular vesicles released during hypoxia transport heparanase and enhance macrophage migration, endothelial tube formation and cancer cell stemness.","authors":"Kaushlendra Tripathi, Shyam K Bandari, Ralph D Sanderson","doi":"10.1002/pgr2.1","DOIUrl":"10.1002/pgr2.1","url":null,"abstract":"<p><p>Heparanase is upregulated during the progression of most cancers and via its enzyme activity promotes extracellular matrix degradation, angiogenesis and cell migration. Heparanase expression is often associated with enhanced tumor aggressiveness and chemoresistance. We previously demonstrated that increased heparanase expression in tumor cells enhances secretion and alters the composition of tumor-released exosomes. In the present study, we discovered that extracellular vesicles (EVs) secreted by human multiple myeloma cells growing in hypoxic conditions exhibited elevated levels of heparanase cargo compared to EVs from cells growing in normoxic conditions. When macrophages (RAW 264.7 monocyte/macrophage-like cells) were exposed to EVs released by tumor cells growing in either hypoxic or normoxic conditions, macrophage migration and invasion was elevated by EVs from hypoxic conditions. The elevated invasion of macrophages was blocked by a monoclonal antibody that inhibits heparanase enzyme activity. Moreover, the heparanase-bearing EVs from hypoxic cells greatly enhanced endothelial cell tube formation consistent with the known role of heparanase in promoting angiogenesis. EVs from hypoxic tumor cells when compared with EVs from normoxic cells also enhanced cancer stemness properties of both CAG and RPMI 8226 human myeloma cells. Together these data indicate that under hypoxic conditions, tumor cells secrete EVs having an elevated level of heparanase as cargo. These EVs can act on both tumor and non-tumor cells, enhancing tumor progression and tumor cell stemness that likely supports chemoresistance and relapse of tumor.</p>","PeriodicalId":74585,"journal":{"name":"Proteoglycan research","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10117102/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9444871","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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