A deleterious mutation in SAMD9 causes normophosphatemic familial tumoral calcinosis

Familial tumoral calcinosis (FTC) is a rare autosomal recessive disorder characterized by the progressive deposition of calcified masses in cutaneous and subcutaneous tissues, which results in painful ulcerative lesions and severe skin and bone infections. Two major types of FTC have been recognized: hyperphosphatemic FTC (HFTC) and normophosphatemic FTC (NFTC). HFTC was recently shown to result from mutations in two different genes: GALNT3, which codes for a glycosyltransferase, and FGF23, which codes for a potent phosphaturic protein. To determine the molecular cause of NFTC, we performed homozygosity mapping in five affected families of Jewish Yemenite origin and mapped NFTC to 7q21-7q21.3. Mutation analysis revealed a homozygous mutation in the SAMD9 gene (K1495E), which was found to segregate with the disease in all families and to interfere with the protein expression. Our data suggest that SAMD9 is involved in the regulation of extraosseous calcification, a process of considerable importance in a wide range of diseases as common as atherosclerosis and autoimmune disorders.     

A new missense mutation in FGF23 gene in a male with hyperostosis–hyperphosphatemia syndrome (HHS)

Hyperostosis–hyperphosphataemia syndrome (HHS) is a rare autosomal recessive metabolic disorder, characterized by recurrent painful swelling of long bones, periosteal new bone formation and cortical hyperostosis or intramedullary sclerosis, hyperphosphatemia and low intact fibroblast growth factor 23 (FGF23) protein levels. It is caused by mutations in 2 genes, N-acetylgalactosaminyltransferase 3 (GalNAc-transferase; GALNT3) and FGF23. We have performed mutation analysis of the GALNT3 and FGF23 genes in a patient with HHS and detected a homozygous mutation in exon 3 of FGF23 gene (NM_020638.2: c.471C>A) which results in amino acid change from phenylalanine 157 to leucin (p.F157L) in receptor interaction site.     

GALNT14与肿瘤

在过去的一个多世纪,许多肿瘤标志物被发现,其中包括多肽N-乙酰半乳糖胺基转移酶(polypeptide N-acetylgalactosaminyltransferase,ppGALNAc-T,简称GALNT)家族中的多个成员。GALNT家族是催化黏蛋白O-糖基化修饰的起始酶,其能够影响黏蛋白的O-糖基化,从而影响肿瘤细胞的发生、预后、增殖与迁移等。GALNT14是该家族中最新发现的成员之一,近年的研究发现,GALNT14在多种肿瘤中表达异常,并与肿瘤细胞的发生、侵袭、转移和凋亡等有关。本文主要对GALNT14蛋白的结构特点及其在肿瘤中的作用进行综述,为进一步研究GALNT14与肿瘤发病机制的关系以及作为潜在的药物靶点提供参考。     

EFEMP2 Mediates GALNT14-Dependent Breast Cancer Cell Invasion

N-Acetylgalactosaminyltransferase-14 (GALNT14) is a member of acetylgalactosaminyltransferases family. We have shown that GALNT14 could promote breast cancer cell invasion. However, the underlying molecular mechanism is unclear. Here, using yeast two hybrid, we find that EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) interacts with GALNT14. Both in vitro and in vivo binding assays show that EFEMP2 is associated with GALNT14. Moreover, we find that GALNT14 mediates glycosylation of EFEMP2. EFEMP2 significantly increased the invasion ability of breast cancer cells including MCF-7 and MBA-MD-231 cells, and this phenomenon is suppressed by knockdown expression of GALNT14. In addition, the GALNT14-dependent O-glycosylation of EFEMP-2 regulates the stability of EFEMP-2 protein in breast cancer cells. Taken together, our results demonstrate a novel molecular mechanism underlying breast cancer invasion.     

GALNT3, a gene associated with hyperphosphatemic familial tumoral calcinosis, is transcriptionally regulated by extracellular phosphate and modulates matrix metalloproteinase activity

GALNT3 encodes UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyl-transferarase 3 (ppGalNacT3), a glycosyltransferase which has been suggested to prevent proteolysis of FGF23, a potent phosphaturic protein. Accordingly, loss-of-function mutations in GALNT3 cause hyperphosphatemic familial tumoral calcinosis (HFTC), a rare autosomal recessive disorder manifesting with increased kidney reabsorption of phosphate, resulting in severe hyperphosphatemia and widespread ectopic calcifications. Although these findings definitely attribute a role to ppGalNacT3 in the regulation of phosphate homeostasis, little is currently known about the factors regulating GALNT3 expression. In addition, the effect of decreased GALNT3 expression in peripheral tissues has not been explored so far. In the present study, we demonstrate that GALNT3 expression is under the regulation of a number of factors known to be associated with phosphate homeostasis, including inorganic phosphate itself, calcium and 1,25-dihydroxyvitamin D(3). In addition, we show that decreased GALNT3 expression in human skin fibroblasts leads to increased expression of FGF7 and of matrix metalloproteinases, which have been previously implicated in the pathogenesis of ectopic calcification. Thus, the present data suggest that ppGalNacT3 may play a role in peripheral tissues of potential relevance to the pathogenesis of disorders of phosphate metabolism.     

GALNT14 Involves the Regulation of Multidrug Resistance in Breast Cancer Cells

GALNT14 is a member of N-acetylgalactosaminyltransferase enzyme family and mediates breast cancer cell development. Here, we find that GALNT14 regulates multidrug resistance (MDR) in breast cancer. The expression of GALNT14 is associated with MDR in breast cancer. Higher level of GALNT14 facilitates MCF-7 cells to resist Adriamycin, whereas knockdown of GALNT14 sensitizes cells to Adriamycin. Moreover, the expression of GALNT14 associates with the expression of P-gp, the efflux pump localized on the cell membrane, which could be the underlying mechanism of how GALNT14 induces MDR. In-depth analysis shows that GALNT14 regulates the stability of P-gp. Finally, GALNT14 associates with higher level of P-gp in chemotherapy-resistant human breast cancer tissues. Taken together, our studies reveal a molecular mechanism in breast cancer MDR.     

MicroRNA-30e Functions as a Tumor Suppressor in Cervical Carcinoma Cells through Targeting GALNT7

Cervical cancer is the third most common cancer in women worldwide. However, the underlying mechanism of occurrence and development of cervical cancer is obscure. In this study, we observed that miR-30e was downregulated in clinical cervical cancer tissues and cervical cancer cells. Next, overexpression of miR-30e reduced the cervical cancer cell growth through MTT, colony formation, EdU, and Transwell assay in SiHa and Caski cells. Subsequently, UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7) was identified as a potential miR-30e target by bioinformatics analysis. Moreover, we showed that miR-30e was able to bind to the 3′UTR of GALNT7 by luciferase reporter assay. In addition, the mRNA and protein levels of GALNT7 in cervical cancer cells were downregulated by miR-30e. And we validated that downregulation of GALNT7 repressed the proliferation of SiHa and Caski cells by MTT, colony formation, and Transwell assay. We identified that the restoration of GALNT7 expression was able to counteract the effect of miR-30e on cell proliferation of cervical cancer cells. Furthermore, we found that the expression levels of GALNT7 were frequently upregulated and negatively correlative to those of miR-30e in cervical cancer tissues. In addition, we validated that restoration of GALNT7 rescued the miR-30e–suppressed growth of cervical cancer xenografts in vivo. In conclusion, the current results suggest that miR-30e may function as tumor suppressors in cervical cancer through downregulation of GALNT7. Both miR-30e and its novel target, GALNT7, may play an important role in the process of cervical cancer.     

GALNT2 Expression Is Reduced in Patients with Type 2 Diabetes: Possible Role of Hyperglycemia

Impaired insulin action plays a major role in the pathogenesis of type 2 diabetes, a chronic metabolic disorder which imposes a tremendous burden to morbidity and mortality worldwide. Unraveling the molecular mechanisms underlying insulin resistance would improve setting up preventive and treatment strategies of type 2 diabetes. Down-regulation of GALNT2, an UDPN-acetyl-alpha-D-galactosamine polypeptideN-acetylgalactosaminyltransferase-2 (ppGalNAc-T2), causes impaired insulin signaling and action in cultured human liver cells. In addition, GALNT2 mRNA levels are down-regulated in liver of spontaneously insulin resistant, diabetic Goto-Kakizaki rats. To investigate the role of GALNT2 in human hyperglycemia, we measured GALNT2 mRNA expression levels in peripheral whole blood cells of 84 non-obese and 46 obese non-diabetic individuals as well as of 98 obese patients with type 2 diabetes. We also measured GALNT2 mRNA expression in human U937 cells cultured under different glucose concentrations. In vivo studies indicated that GALNT2 mRNA levels were significantly reduced from non obese control to obese non diabetic and to obese diabetic individuals (p<0.001). In vitro studies showed that GALNT2 mRNA levels was reduced in U937 cells exposed to high glucose concentrations (i.e. 25 mmol/l glucose) as compared to cells exposed to low glucose concentration (i.e. 5.5 mmol/l glucose +19.5 mmol/l mannitol). In conclusion, our data indicate that GALNT2 is down-regulated in patients with type 2 diabetes and suggest that this association is, at least partly, secondary to hyperglycemia. Further studies are needed to understand whether GALNT2 down-regulation plays a pathogenic role in maintaining and/or aggravating the metabolic abnormalities of diabetic milieu.     

Mutations in B3GALNT2 Cause Congenital Muscular Dystrophy and Hypoglycosylation of α-Dystroglycan

Mutations in several known or putative glycosyltransferases cause glycosylation defects in α-dystroglycan (α-DG), an integral component of the dystrophin glycoprotein complex. The hypoglycosylation reduces the ability of α-DG to bind laminin and other extracellular matrix ligands and is responsible for the pathogenesis of an inherited subset of muscular dystrophies known as the dystroglycanopathies. By exome and Sanger sequencing we identified two individuals affected by a dystroglycanopathy with mutations in β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2). B3GALNT2 transfers N-acetyl galactosamine (GalNAc) in a β-1,3 linkage to N-acetyl glucosamine (GlcNAc). A subsequent study of a separate cohort of individuals identified recessive mutations in four additional cases that were all affected by dystroglycanopathy with structural brain involvement. We show that functional dystroglycan glycosylation was reduced in the fibroblasts and muscle (when available) of these individuals via flow cytometry, immunoblotting, and immunocytochemistry. B3GALNT2 localized to the endoplasmic reticulum, and this localization was perturbed by some of the missense mutations identified. Moreover, knockdown of b3galnt2 in zebrafish recapitulated the human congenital muscular dystrophy phenotype with reduced motility, brain abnormalities, and disordered muscle fibers with evidence of damage to both the myosepta and the sarcolemma. Functional dystroglycan glycosylation was also reduced in the b3galnt2 knockdown zebrafish embryos. Together these results demonstrate a role for B3GALNT2 in the glycosylation of α-DG and show that B3GALNT2 mutations can cause dystroglycanopathy with muscle and brain involvement.     

Role of GALNT4 in protecting against cardiac hypertrophy through ASK1 signaling pathway

Pathological myocardial hypertrophy is regulated by multiple pathways. However, its underlying pathogenesis has not been fully explored. The goal of this work was to elucidate the function of polypeptide N-acetylgalactosaminyltransferase 4 (GALNT4) in myocardial hypertrophy and its underlying mechanism of action. We illustrated that GALNT4 was upregulated in the models of hypertrophy. Two cardiac hypertrophy models were established through partial transection of the aorta in GALNT4-knockout (GALNT4-KO) mice and adeno-associated virus 9-GALNT4 (AAV9-GALNT4) mice. The GALNT4-KO mice demonstrated accelerated cardiac hypertrophy, dysfunction, and fibrosis, whereas the opposite phenotype was observed in AAV9-GALNT4 mice. Similarly, GALNT4 overexpression mitigated the degree of phenylephrine-induced cardiomyocyte hypertrophy in vitro whereas GALNT4 knockdown aggravated the hypertrophy. In terms of mechanism, GALNT4 deficiency increased the phosphorylation and activation of ASK1 and its downstream targets (JNK and p38), whereas GALNT4 overexpression inhibited activation of the ASK1 pathway. Furthermore, we demonstrated that GALNT4 can directly bind to ASK1 inhibiting its N-terminally mediated dimerization and the subsequent phosphorylation of ASK1. Finally, an ASK1 inhibitor (iASK1) was able to reverse the effects of GALNT4 in vitro. In summary, GALNT4 may serve as a new regulatory factor and therapeutic target by blocking the activation of the ASK1 signaling cascade.Subject terms: Protein-protein interaction networks, Heart failure     

重组人GALNT3-sol蛋白在毕赤酵母中的高效表达和活性鉴定

目的:为了获得有催化活性的人乙酰半乳糖胺转移酶3(GALNT3),构建了GALNT3可溶性区域(GALNT3-sol)的真核分泌表达载体,在巴斯德毕赤酵母中表达并纯化GALNT3-sol蛋白,体外检测其转糖基活性。方法:以构建好的pET15b/GALNT3-sol为模板进行PCR,扩增编码人GALNT3-sol的cDNA片段(1 755 bp),将其克隆至真核表达载体pPIC9K,载体线性化后采用电击法转化毕赤酵母GS115。通过MD平板和G418平板筛选出阳性高拷贝重组菌株。阳性菌株经过甲醇诱导表达人GALNT3-sol重组蛋白,表达上清进行Ni-NAT分离纯化。分别采用SDS-PAGE和Western blot鉴定纯化的重组蛋白,并使用HPLC和MALDI-TOF/MS分析其转糖基化反应的活性。结果:成功构建了能够分泌表达GALNT3-sol的毕赤酵母菌株。阳性表达菌株在BMMY培养基(pH 6.0)中20℃培养,经0.5%甲醇诱导表达96 h,摇瓶表达量可达5mg/L。SDS-PAGE和Western blot结果显示表达重组蛋白为糖基化形式。活性检测显示表达的重组蛋白具有转糖基活性。结论:成功获得可以高效分泌表达具有活性的人GALNT3-sol蛋白的毕赤酵母菌株,为进一步研究人GALNT3的性质及其应用提供了基础。     

Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N- acetylgalactosaminyltransferase family

A homologous family of UDP- N -acetylgalactosamine: polypeptide N - acetylgalactosaminyltransferases (GalNAc-transferases) initiate O- glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc- transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.      

Generalist predation on forest tent caterpillar varies with forest stand composition: an experimental study across multiple life stages

1. Generalist enemies can regulate low‐density forest insect populations, and are widely considered to cause greater mortality in more diverse habitats. Forest tent caterpillars (Malacosoma disstria Hübner; FTC) are a major defoliator of aspen (Populus tremuloides Micheaux) in the boreal forest, a region with a mosaic of forest stand types. This heterogeneity may influence FTC outbreaks if generalist predation or parasitism differs among stands of different tree composition. 2. Using exclusion experiments we estimate predation and parasitism of FTC across multiple life‐history stages in low‐density populations occupying both aspen (low diversity) and mixedwood stands (high diversity). 3. Arthropod and avian generalist predators were responsible for most natural enemy‐caused mortality of immature FTC, but their relative impacts varied among FTC life‐history stages. Contrary to expectation, predation on late instar larvae and pupae was higher in the less diverse aspen stands and early instar mortality did not differ. 4. By considering multiple life‐history stages, our results provide a more comprehensive view of natural enemy‐caused morality of immature FTC. Because generalist predation on FTC was higher in aspen than in mixedwood stands, we suggest that FTC populations may be slower to reach outbreak levels in aspen stands.     

蛙下丘频率调谐特性方向选择性的建模和仿真

基于Ｆｅｎｇ等人的神经电生理实验结果,建立了关于豹蛙（Ｒａｎａ ｐｉｐｉｅｎ）半规隆凸（ｔｏｒｕｓ ｓｅｍｉｃｉｒｃｕｌａｒｉｓ）核团神经元频率调谐特性方向选择性反应的模型,并在此基础上讨论了模型各个参数对模型方向性反应输出的影响。结果提示：１）听觉系统两侧的相互抑制作用－双耳抑制主要决定频率调谐特性的尖锐性随方向变化的特性;２）前级神经元对不同方位外界声源刺激具有不同强度平均脉冲发放的特征则主要     

Segregation of LIPG, CETP, and GALNT2 Mutations in Caucasian Families with Extremely High HDL Cholesterol

To date, few mutations are described to underlie highly-elevated HDLc levels in families. Here we sequenced the coding regions and adjacent sequence of the LIPG, CETP, and GALNT2 genes in 171 unrelated Dutch Caucasian probands with HDLc≥90th percentile and analyzed segregation of mutations with lipid phenotypes in family members. In these probands, mutations were most frequent in LIPG (12.9%) followed by GALNT2 (2.3%) and CETP (0.6%). A total of 6 of 10 mutations in these three genes were novel (60.0%), and mutations segregated with elevated HDLc in families. Interestingly, the LIPG mutations N396S and R476W, which usually result in elevated HDLc, were unexpectedly found in 6 probands with low HDLc (i.e., ≤10th percentile). However, 5 of these probands also carried mutations in ABCA1, LCAT, or LPL. Finally, no CETP and GALNT2 mutations were found in 136 unrelated probands with low HDLc. Taken together, we show that rare coding and splicing mutations in LIPG, CETP, and GALNT2 are enriched in persons with hyperalphalipoproteinemia and segregate with elevated HDLc in families. Moreover, LIPG mutations do not overcome low HDLc in individuals with ABCA1 and possibly LCAT and LPL mutations, indicating that LIPG affects HDLc levels downstream of these proteins.