Xylose phosphorylation functions as a molecular switch to regulate proteoglycan biosynthesis

Most eukaryotic cells elaborate several proteoglycans critical for transmitting biochemical signals into and between cells. However, the regulation of proteoglycan biosynthesis is not completely understood. We show that the atypical secretory kinase family with sequence similarity 20, member B (Fam20B) phosphorylates the initiating xylose residue in the proteoglycan tetrasaccharide linkage region, and that this event functions as a molecular switch to regulate subsequent glycosaminoglycan assembly. Proteoglycans from FAM20B knockout cells contain a truncated tetrasaccharide linkage region consisting of a disaccharide capped with sialic acid (Siaα2–3Galβ1–4Xylβ1) that cannot be further elongated. We also show that the activity of galactosyl transferase II (GalT-II, B3GalT6), a key enzyme in the biosynthesis of the tetrasaccharide linkage region, is dramatically increased by Fam20B-dependent xylose phosphorylation. Inactivating mutations in the GALT-II gene (B3GALT6) associated with Ehlers-Danlos syndrome cause proteoglycan maturation defects similar to FAM20B deletion. Collectively, our findings suggest that GalT-II function is impaired by loss of Fam20B-dependent xylose phosphorylation and reveal a previously unappreciated mechanism for regulation of proteoglycan biosynthesis.The human genome encodes more than 500 protein kinases, most of which phosphorylate protein substrates in the nucleus and cytosol and play important roles in cell signaling (1, 2). Kinases that specifically phosphorylate glycans have only rarely been reported and our knowledge of their physiological functions remains in its infancy (3–5). Fam20B (family with sequence similarity 20, member B) is a recently identified atypical secretory pathway kinase (6, 7). Genetic studies have shown that deletion of Fam20B in mice results in embryonic lethality at embryonic day 13.5 (E13.5), whereas loss-of-function mutations in the fam20b gene in Danio rerio cause aberrant cartilage formation and severe skeletal defects that are linked to abnormal proteoglycan (PG) biosynthesis (8, 9).PGs are a special family of glycoconjugates consisting of one or more glycosaminoglycan (GAG) side chains covalently linked to specific Ser residues within a protein core via a common linkage tetrasaccharide [glucuronic acid-β1–3-galactose-β1–3-galactose-β1–4-xylose-β1 (GlcAβ1–3Galβ1–3Galβ1–4Xylβ1)] as illustrated in Fig. 1A (10). Mature GAGs are sulfated linear polysaccharides that are further classified as heparan sulfate (HS) or chondroitin/dermatan sulfate (CS), depending on the specific composition of elongated sugar repeats (11). PGs are expressed on the cell surface and in the extracellular matrix of all animal cells and tissues, playing critical roles in cell–cell and cell–matrix interactions and signaling, largely through the attached GAGs (12). The cellular machinery required for PG biosynthesis is conserved over a wide range of eukaryotic organisms and altered PG biosynthesis is associated with numerous human disease states (13–17). Thus, understanding how this process is regulated will be important. Loss of Fam20B appears to decrease the amount of cellular GAG chains and causes profound defects in embryonic development and skeletal formation (6, 8, 9). Fam20B has been reported to have xylose kinase activity against α-thrombomodulin, a glycoprotein bearing the tetrasaccharide linkage fragment. However, the specific molecular mechanism by which Fam20B-dependent xylose phosphorylation regulates PG synthesis is not clear.Open in a separate windowFig. 1.Xylosyl kinase Fam20B phosphorylates authentic proteoglycans. (A) Schematic representation of proteoglycan core proteins, glycosaminoglycan side chains, structure of the linkage tetrasaccharide, and biosynthetic enzymes involved. (B) Gel electrophoresis and Coomassie staining of recombinant Fam20B protein purified from insect cell conditioned medium. (C) Time-dependent incorporation of ³²P from [γ-³²P]ATP into decorin by Fam20B or Fam20B D309A (D/A) treated with or without chondroitinase ABC (Chon-ABC). Reaction products were analyzed by gel electrophoresis and autoradiography (autorad). (D) Lentiviral ShRNA mediated knockdown of Fam20B in MRC-5 cells and [³²P]orthophosphate metabolic labeling. Fam20B knockdown efficiency was determined by immunoblotting of endogeneous Fam20B. The level of decorin phosphorylation in the control (Sh-ctrl) and Fam20B knockdown (ShRNA) cells were visualized by ³²P autoradiography after decorin immunoprecipitation and gel electrophoresis. (E) Structure of synthetic Tetra-Ben as a model substrate for Fam20B. (F) Fam20B kinase reaction velocity versus [Mn²⁺]ATP concentration using Tetra-Ben as a substrate. (G) Fam20B kinase reaction velocities versus concentration of Tetra-Ben, Gal-Xyl-Ben, and Xyl-Ben artificial substrates. The reaction products for F and G were purified using SepPak C18 cartridges and the incorporated ³²P radioactivity was quantified by scintillation counting. Data points were fitted by nonlinear regression of the Michaelis–Menten equation. Error bars are SD of three independent experiments.Here we provide evidence that Fam20B is a xylose kinase that phosphorylates the initiator xylose residue within the tetrasaccharide linkage region of a wide array of O-linked PGs. We show that Fam20B requires a minimal Gal-Xyl disaccharide motif for activity, and loss of Fam20B-dependent xylose phosphorylation results in premature termination of the tetrasaccharide linkage and impaired glycosaminoglycan assembly. Our findings suggest that phosphorylation of xylose within the linkage region by Fam20B dramatically increases the activity of galactosyltransferase II (GalT-II), an enzyme necessary for completion of the linkage region and efficient glycosaminoglycan assembly. Cells lacking either Fam20B or GalT-II are unable to complete the assembly of the linkage region and exhibit only short sugar stubs within their proteoglycans. Our findings reveal a previously unappreciated mechanism for regulation of proteoglycan assembly and offer insight into the molecular basis of diseases associated with aberrant proteoglycan maturation.     

Microvessel density and vascular basement membrane immunostaining in tumours of the breast

There is a well established correlation between increased breast tumour microvessel density (MVD) and reduced prognosis. The aims of this study were to investigate (1) if MVD is elevated in regions other than `hotspots' of node positive versus node negative breast tumours, and (2) to quantitate the percentage of vessels without vascular basement membrane (VBM) components in high vascular density (HVD) and average vascular density (AVD) regions of node positive and node negative breast tumours. Serial sections were immunostained for CD31 and double-stained for CD31 and collagen IV (CollIV), laminin (LAM) or heparan sulphate proteoglycan (HSPG). Microvessel counts were obtained from HVD and AVD regions and the number of VBM positive vessels were expressed as a percentage of total CD31 positive vessels. MVD was significantly higher in both the HVD and AVD regions of node positive compared with node negative breast tumours (t-test; P < 0.03). The average percent vessels positive for CollIV, LAM or HSPG ranged from 18%–45% and did not differ between node positive and negative breast tumours (t-test; P > 0.05). No differences were observed in VBM immunostaining between regions of HVD and AVD (t-test; P > 0.05). These results demonstrate that vascular density is elevated throughout node positive breast tumours, rather than just in `hotspots', and show that there is no apparent difference in the percentage of VBM-naked vessels in node positive versus node negative breast tumours. This revised version was published online in June 2006 with corrections to the Cover Date.     

The absence of the embryo in the pseudopregnant uterus alters the deposition of some ECM molecules during decidualization in mice

The embryo-implantation promotes deep changes in the uterus resulting in the formation of a new structure at the maternal–fetal interface, the decidua. Decidualization can also be induced in pseudopregnant rodents resulting in a structure called deciduoma that is morphologically and functionally similar to the decidua. Previous studies from our and other laboratories demonstrate that in rodents, decidualization of the endometrium requires remarkable remodeling of the endometrial extracellular matrix (ECM) that is mainly coordinated by estradiol and progesterone. The influence of the embryo in this process, however, has not yet been investigated. To enlarge the knowledge on this subject, the present study investigates the behavior of a set of ECM molecules, in the absence of paracrine cues originated from the embryo. For that deciduoma was induced in pseudopregnant Swiss mice, and the distribution of collagen types I, III, IV, V and the proteoglycans decorin and biglycan was investigated by immunolabeling from the fifth to the eighth day of pseudopregnancy. It was observed the deposition of collagen types III and IV as well as decorin and biglycan was similar to that previously described by our group in the decidua. However, in the absence of the embryo, some differences occur in the distribution of collagen types I and V, suggesting that beside the major role of ovarian hormones on the endometrial ECM remodeling, molecular signals originated from the conceptus may influence this process.     

动脉粥样硬化高低发区年轻人冠状动脉硫酸软骨素蛋白聚糖的比较研究

选取动脉粥样硬化高发区（北京）１９例、低发区（宁波）１３例年轻人冠状动脉，行硫酸软骨素蛋白聚糖免疫组织化学染色，应用图象分析系统对切片中硫酸软骨素蛋白聚糖行定量分析及比较。１例心脏标本的冠状动脉行硫酸软骨素蛋白聚糖免疫电镜染色。观察分析结果为：①硫酸软骨素蛋白聚糖阳性物质是丝网状分布于细胞外间质中，内膜中硫酸软骨素蛋白聚糖含量高于中膜；②胶体金颗粒多分布于细胞外间质的疏松区，少量贴附于胶原纤维上；③北京地区年轻人冠状动脉内硫酸软骨素蛋白聚糖含量高于宁波地区。提示硫酸软骨素蛋白聚糖在冠状动脉内含量差异可能与高、低发区冠状动脉粥样硬化发病率不同有关。     

hSulf1 Sulfatase promotes apoptosis of hepatocellular cancer cells by decreasing heparin-binding growth factor signaling

: The heparin-binding growth factors fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) are potent mitogens for hepatocellular carcinomas (HCCs). Heparin-binding growth factor signaling is regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPGs). We hypothesized that hSulf1, a recently described sulfatase, regulates growth signaling in HCCs. :Expression of hSulf1 in human HCC tumors was determined by real-time PCR. Down-regulation of hSulf1 expression was investigated by analyzing loss of heterozygosity (LOH) at the hSulf1 locus and the effect of the DNA methylation inhibitor 5-aza-deoxycytidine on hSulf1 expression. The subcellular location of hSulf1 and sulfation state of cell-surface HSPGs were assessed by immunocytochemistry. FGF and HGF signaling was examined by phospho-specific immunoblot analysis. Cell growth was measured by trypan blue exclusion, and the MTT assay and apoptosis were quantitated by fluorescence microscopy. :hSulf1 expression was decreased in 29% of HCCs and 82% of HCC cell lines. There was LOH at the hSulf1 locus in 42% of HCCs. Treatment with 5-aza-deoxycytidine reactivated hSulf1 expression in hSulf1-negative cell lines. Low hSulf1-expressing cells showed increased sulfation of cell-surface HSPGs, enhanced FGF and HGF-mediated signaling, and increased HCC cell growth. Conversely, forced expression of hSulf1 decreased sulfation of cell-surface HSPGs and abrogated growth signaling. HCC cells with high-level hSulf1 expression were sensitive to staurosporine- or cisplatin-induced apoptosis, whereas low expressing cells were resistant. Transfection of hSulf1 into hSulf1-negative cells restored staurosporine and cisplatin sensitivity. :Down-regulation of hSulf1 contributes to hepatocarcinogenesis by enhancing heparin-binding growth factor signaling and resistance to apoptosis.     

Connective tissue growth factor (CCN2) in rat pancreatic stellate cell function: integrin alpha5beta1 as a novel CCN2 receptor

BACKGROUND & AIMS: Pancreatic stellate cells (PSCs) are proposed to play a key role in the development of pancreatic fibrosis. The aim of this study was to evaluate the production by rat activated PSCs of the fibrogenic protein, connective tissue growth factor (CCN2), and to determine the effects of CCN2 on PSC function. METHODS: CCN2 production was evaluated by immunoprecipitation and promoter activity assays. Expression of integrin alpha5beta1 was examined by immunoprecipitation and Western blot. Binding between CCN2 and integrin alpha5beta1 was determined in cell-free systems. CCN2 was assessed for its stimulation of PSC adhesion, migration, proliferation, DNA synthesis, and collagen I synthesis. RESULTS: CCN2 was produced by activated PSCs, and its levels were enhanced by transforming growth factor beta1 treatment. CCN2 promoter activity was stimulated by transforming growth factor beta1, platelet-derived growth factor, alcohol, or acetaldehyde. CCN2 stimulated integrin alpha5beta1-dependent adhesion, migration, and collagen I synthesis in PSCs. Integrin alpha5beta1 production by PSCs was verified by immunoprecipitation, while direct binding between integrin alpha5beta1 and CCN2 was confirmed in cell-free binding assays. Cell surface heparan sulfate proteoglycans functioned as a partner of integrin alpha5beta1 in regulating adhesion of PSCs to CCN2. PSC proliferation and DNA synthesis were enhanced by CCN2. CONCLUSIONS: PSCs synthesize CCN2 during activation and after stimulation by profibrogenic molecules. CCN2 regulates PSC function via cell surface integrin alpha5beta1 and heparan sulfate proteoglycan receptors. These data support a role for CCN2 in PSC-mediated fibrogenesis and highlight CCN2 and its receptors as potential novel therapeutic targets.     

胰腺癌组织Syndecan-1与E-cadherin表达及意义

目的探讨Syndecan-1和E-cadherin在胰腺癌组织中的表达及其临床意义。方法采用免疫组织化学PV6000法检测47例胰腺癌组织和10例正常胰腺组织中Syndecan-1及E-cadherin的表达。结果 10例正常胰腺组织中Syndecan-1均呈阴性表达,而E-cadherin均为阳性表达。胰腺癌组织中Syndecan-1和E-cadherin的表达阳性率分别为44.7%和46.8%,二者均与胰腺癌的分化程度、有无淋巴结转移和临床分期有关(χ2=6.01~9.75,P<0.05),与病人性别、年龄、肿瘤大小和1年生存期无关(χ2=0.28~3.63,P>0.05)。结论 Syndecan-1和E-cadherin在胰腺癌的发生发展中起重要作用,其表达水平可作为判断胰腺癌生物学行为的参考指标。     

人白细胞DR抗原在冠状动脉粥样硬化早期病变中的表达及与CS-PG的关系

为研究人白细胞ＤＲ抗原和硫酸软骨素蛋白聚糖在人冠状动脉中的表达，将１３例年龄人的冠状动脉用福尔马林固定后，进行石蜡包埋，其中６例为对照组，７例为病变组，连续切片后，应用微波炉对两组各例进行ＨＬＡ－ＤＲ和ＣＳ－ＰＧ免疫组织化学染色。切片经图象分析仪定量后，所得结果显示，病变组内膜ＨＬＡ－ＤＲ阳性细胞数密度和ＣＳ－ＰＧ阳性细胞面密度都分别明显高于对照组（Ｐ〈０．０１），并且对照组和病变组内膜ＨＬＡ－Ｄ     

Engineering zonal cartilaginous tissue by modulating oxygen levels and mechanical cues through the depth of infrapatellar fat pad stem cell laden hydrogels

Engineering tissues with a structure and spatial composition mimicking those of native articular cartilage (AC) remains a challenge. This study examined if infrapatellar fat pad‐derived stem cells (FPSCs) can be used to engineer cartilage grafts with a bulk composition and a spatial distribution of matrix similar to the native tissue. In an attempt to mimic the oxygen gradients and mechanical environment within AC, FPSC‐laden hydrogels (either 2 mm or 4 mm in height) were confined to half of their thickness and/or subjected to dynamic compression (DC). Confining FPSC‐laden hydrogels was predicted to accentuate the gradient in oxygen tension through the depth of the constructs (higher in the top and lower in the bottom), leading to enhanced glycosaminoglycan (GAG) and collagen synthesis in 2 mm high tissues. When subjected to DC alone, both GAG and collagen accumulation increased within 2 mm high unconfined constructs. Furthermore, the dynamic modulus of constructs increased from 0.96 MPa to 1.45 MPa following the application of DC. There was no synergistic benefit of coupling confinement and DC on overall levels of matrix accumulation; however in all constructs, irrespective of their height, the combination of these boundary conditions led to the development of engineered tissues that spatially best resembled native AC. The superficial region of these constructs mimicked that of native tissue, staining weakly for GAG, strongly for type II collagen, and in 4 mm high tissues more intensely for proteoglycan 4 (lubricin). This study demonstrated that FPSCs respond to joint‐like environmental conditions by producing cartilage tissues mimicking native AC. Copyright © 2016 John Wiley & Sons, Ltd.     

CSPG4在胃肠间质瘤中的表达及其与临床参数及肿瘤免疫细胞的关系

目的:通过检测硫酸软骨素蛋白聚糖4(chondroitin sulfate proteoglycan 4，CSPG4)在胃肠间质瘤（gastrointestinal stromal tumor，GIST）中的表达情况,探讨其与临床病理参数和免疫细胞的相关性。方法:运用免疫组化EnVision二步法检测162例GIST中CSPG4蛋白的表达情况，分析其与年龄、性别、发生部位及NIH危险度分级的相关性，并探讨GIST中CSPG4蛋白的表达与肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes，TILs）CD4+、CD8+、CD20+、CD56+及CD68+细胞计数的关系。结果:CSPG4蛋白在GIST中的表达与发生部位及NIH危险度分级相关(P＜0.05)，与年龄及性别差异无统计学意义(P＞0.05)。CSPG4蛋白在GIST中的表达与CD8+、CD56+及CD68+细胞计数相关(P＜0.05)，而与CD4+及CD20+细胞计数差异无统计学意义(P＞0.05)。结论:CSPG4蛋白表达水平在一定程度上可预测GIST的生物学行为，CSPG4蛋白可能通过调节肿瘤免疫微环境参与GIST的发生和发展。