Journal: Cell Proliferation

Article Title: Glycomics reveal that ST6GAL1‐mediated sialylation regulates uterine lumen closure during implantation

doi: 10.1111/cpr.13169

Figure Lengend Snippet: Expression patterns of the sialylated glycans as well as the mRNA and protein expression of ST6GAL1 and ST6GAL2 in pig endometrium during implantation. (A) The 19 possible α2,3/6‐sialylated N‐glycans identified by MALDI‐TOF MS. Fucose ; N‐acetylglucosamine ; N‐acetyl neuraminic acid ; N‐glycolyl neuraminic acid ; mannose ; Galactose . (B) Representative images of lectin fluorescence assays taken from pig endometrium on gestational days 12, 15 and 18 (n = 3 gilts/gestational day). MAL‐II was used to detect α2,3‐linked sialic acid residues. SNA was used to detect α2,6‐linked sialic acid residues. (C) Expression levels of ST6GAL1 and ST6GAL2 in pig endometrium on gestational days 12, 15 and 18 measured by qRT‐PCR (n = 3 gilts/gestational day). The error bars represent the standard error. Mean is denoted by a red line. Nonparametric Mann‐Whitney one tail test was used for statistical analysis. (D) Distributions of ST6GAL1 and ST6GAL2 in pig endometrium on gestational days 12, 15 and 18 (n = 3 gilts/gestational day). The positive signal is in green, while the nucleus is in blue. NC, negative control. LE, endometrial luminal epithelium. GD, gestational day. Scale bar = 50 µm

Article Snippet: The primary antibodies used were ST6GAL1 (1:50, ab77676, abcam), ST6GAL2 (1:50, AF7747; R&D Systems), E‐cadherin (1:50, ab40772; abcam), β‐catenin (1:50, ab6302; abcam), Vimentin (1:50; sc‐73258), Rac1 (1:30, 66122–1‐Ig; Proteintech) and RhoA (1:50, 10749–1‐Ig; Proteintech).

Techniques: Expressing, Fluorescence, Quantitative RT-PCR, MANN-WHITNEY, Negative Control

Journal: Orthopaedic Journal of Sports Medicine

Article Title: Clinical Outcome 3 Years After Autologous Chondrocyte Implantation Does Not Correlate With the Expression of a Predefined Gene Marker Set in Chondrocytes Prior to Implantation but Is Associated With Critical Signaling Pathways

doi: 10.1177/2325967114550781

Figure Lengend Snippet: Induced Genes in the Success Group

Article Snippet: The genes analyzed with qPCR were selected from the predefined marker set and from genes annotated with enriched biological process terms in the gene ontology (GO) analysis (assay number in parentheses): COL2A1 (Hs00156568_m1), BMP2 (Hs00154192_m1), FGFR3 (Hs00179829_m1), ALK1 (Hs00163543_m1), ACAN (Hs00153936_m1), CD44 (Hs01075862_m1), PTPRD (Hs00369913_m1), PTPRF (Hs00892965_m1), ST6GAL2 (Hs00383641_m1), GCLC (Hs00155249_m1), PRLR (Hs01061477_m1), GRIK2 (Hs00222637_m1), LYST (Hs00915897_m1), AGT (Hs01586213_m1), FAIM2 (Hs00392342_m1), PHLDA1 (Hs00378285_g1).

Techniques: Protein Binding

Journal: Nature Communications

Article Title: Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis

doi: 10.1038/s41467-021-22365-z

Figure Lengend Snippet: a Illustration shows pathways involved in sialic acid and sialylated glycans biosynthesis. Relative expression for each gene was extracted from bulk RNA-Seq data comparing naive and arthritic SFs directly isolated from mouse synovium. b Synovial fibroblasts expanded from healthy or arthritic mouse joints were stimulated in vitro for 6 h with recombinant IL-1β, TNF, or IL-17A [10 ng/ml]. mRNA expression for genes involved in sialylation [as shown in a ] was evaluated by RT-qPCR. Il6 was included as a positive control to confirm cell activation. Volcano plots show log2 fold difference in stimulated cells ( x axis) and p -value ( y axis). Each dot represents the mean of three independent experiments analyzed in triplicate, with naive cells in blue and arthritic cells in red. The pink line indicates a twofold-change in gene expression threshold and blue lines indicate p = 0.05 threshold in the t -test to evaluate statistical significance. c TNF, but not IL-1β or IL-17A reduces α2-6-sialylation of synovial fibroblasts both at RNA ( St6gal1 expression) and protein (SNA binding) levels. Expression of St6gal1 (left panel ) was evaluated by RT-qPCR. Results show the mean of three independent experiments analyzed in triplicate, error bars represent SEM. Statistical significance was determined using two-tail unpaired t -tests where * p < 0.05 and ** p < 0.01. Actual p -values: a 0.0069, b 0.0277. Expression of α2-6 sialylated glycoconjugates was determined by SNA binding (right panel) as in Fig. . Results are merged from 3 (naive) and 2 (CIA) independent experiments, n = 10 (DMEM naive), 16 (IL-1 naive and IL-17 naive), 13 (TNF naive), 10 (DMEM CIA), 12 (IL-1 naive and IL-17 naive), and 9 (TNF CIA). Statistical significance was determined using two-tail unpaired t -tests, where ** p < 0.01 and *** p < 0.001. Actual p -values: c 0.0124, d 0.0091. d SFs were expanded from OA human synovium. Cells were stimulated with recombinant human TNF (10 ng/ml) for 6 h. IL6 , ST6GAL1, and ST6GAL2 expression were determined by qPCR. Results show relative expression to HPRT, showing mean ± SEM. Statistics: one-tail unpaired t -test, n = 3 biological replicates from cells pooled from three donors, *** p < 0.001, * p < 0.05. Actual p -values: e <0.0001, f 0.0170, g 0.0321. e Relative expression of individual sialylated glycan structures was evaluated by MALDI-TOF MS analysis as in Fig. . Ratios of sialylated vs non-sialylated twin structures were evaluated for non-stimulated cells (blue) and TNF stimulated (red) cells (48 h, 10 ng/ml). Results are from one single experiment using pooled cells from three animals.

Article Snippet: TaqMan predesigned probes (ThermoFisher Scientific) were used to evaluate mouse Actb (4352933E), Il6 (Mm00446190_m1 ), St6gal1 (Mn00486119_m1) , St6gal2( Mm01268915_m1) , St3gal1 (Mm00501493_m1) , St6galnac1 (Mm01252949_m1) , St6galnac3 (Mm01316813_m1) and Mmp3 (Mm00440295_m1) and human IL6( Hs00174131_m1) , ST6GAL1 (Hs00949382_m1) , ST6GAL2 (Hs00383641) and HPRT (4333768T).

Techniques: Expressing, RNA Sequencing, Isolation, In Vitro, Recombinant, Quantitative RT-PCR, Positive Control, Activation Assay, Gene Expression, Binding Assay, Glycoproteomics

Journal: The Journal of Biological Chemistry

Article Title: Molecular Phylogeny and Functional Genomics of ?-Galactoside ?2,6-Sialyltransferases That Explain Ubiquitous Expression of st6gal1 Gene in Amniotes *

doi: 10.1074/jbc.M110.163931

Figure Lengend Snippet: A, ML phylogenetic tree of 36 sialyltransferases of the ST6Gal family. A maximum likelihood phylogenetic tree was inferred by the Phyml, JTT model of amino acid substitution: 36 ST6Gal sequences, 11 vertebrate ST6Gal I, 11 vertebrate ST6Gal II. G-BLOCKS selected 200 positions. Bootstrap values were calculated from 500 replicates, and values of >50% are reported at the left of each divergence point. The tree is rooted with the invertebrate arthropod sequences as the outgroup. B, calculation of divergence time between ST6Gal I and ST6Gal II. The lengths of each horizontal branch were calculated from the linearized tree obtained with ME (Min. Evol.) using MEGA4.0.

Article Snippet: In vertebrates, most examined genomes contain two members the st6gal1 and st6gal2 paralogous genes, except in the lamprey Petromyzon marinus , where three st6gal copies were found.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Molecular Phylogeny and Functional Genomics of ?-Galactoside ?2,6-Sialyltransferases That Explain Ubiquitous Expression of st6gal1 Gene in Amniotes *

doi: 10.1074/jbc.M110.163931

Figure Lengend Snippet: Indel insertion/deletion events in the ST6Gal I and ST6Gal II protein sequences. A, alignments of protein sequences without gap are schematized in gray; indels are indicated by white boxes. Intron insertions are indicated by black bars in the teleost st6gal1 sequence. B, simplified phylogenetic tree and insertion/deletion distribution. The tree was constructed with 200 positions within the catalytic domain, using ME. Filled square, insertion; open squares, indel; filled rectangles, intron insertion; open triangles, deletion. C, correlation between Indel numbers and branch lengths. The tree was constructed as in B. The regression equation was given by PAST 2.01.

Article Snippet: In vertebrates, most examined genomes contain two members the st6gal1 and st6gal2 paralogous genes, except in the lamprey Petromyzon marinus , where three st6gal copies were found.

Techniques: Sequencing, Construct

Journal: The Journal of Biological Chemistry

Article Title: Molecular Phylogeny and Functional Genomics of ?-Galactoside ?2,6-Sialyltransferases That Explain Ubiquitous Expression of st6gal1 Gene in Amniotes *

doi: 10.1074/jbc.M110.163931

Figure Lengend Snippet: Genomic organization of human st6gal1/st6gal2 cluster paralogon and putative orthologous counterpart is indicative of a WGD event. Eleven putative paralogous genes spanning regions on human chromosomes HSA2q11.3 and HSA3q27 were found. Genes represented were chosen from analysis performed at Ensembl by chromosomal walking and reciprocal TBLASTN searches of genes adjacent to st6gal loci.

Article Snippet: In vertebrates, most examined genomes contain two members the st6gal1 and st6gal2 paralogous genes, except in the lamprey Petromyzon marinus , where three st6gal copies were found.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Molecular Phylogeny and Functional Genomics of ?-Galactoside ?2,6-Sialyltransferases That Explain Ubiquitous Expression of st6gal1 Gene in Amniotes *

doi: 10.1074/jbc.M110.163931

Figure Lengend Snippet: Syntenic relationships of the st6gal gene loci in vertebrate and amphioxus genomes. Physically mapped genomes of human (HSA), mouse (MMU), chicken (GGA), O. latipes (OLA), T. nigroviridis (TNI), G. aculeatus (GAC), and D. rerio (DRE) in Ensembl were used to identify conserved gene neighbors of the st6gal genes. A, conserved syntenic blocks found around the st6gal2 gene; B, disrupted synteny around the st6gal1 gene found in amniotes and in teleost genomes. C, schematic diagram depicting the genetic events that occurred early in the jawed vertebrate lineage and led to modern st6gal genes in fish. The last common ancestor (LCA) of teleost fishes and amniotes around 500 MYA bore an ancestral st6gal gene designated the st6gal1/2 gene, which was located on the protochromosome DRE 15-2-6-9 HSA 2-3; WGD R2 (∼475 MYA) gave rise to st6gal1 and st6gal2 genes found on HSA3 and HSA2, respectively, and on the protochromosome DRE 15-2 and DRE 6-9 of the teleost last common ancestor. The DRE 2/DRE 15 and DRE 6/DRE 9 pairs of chromosomes derived from these single common protochromosomes DRE 15-2 and DRE 6-9 after WGD R3. Rearrangement of a block of genes including st6gal1 occurred in teleosts, resulting in the translocation of st6gal1 on DRE21, whereas the other duplicate, named st6gal1-r, disappeared from DRE 15 through pseudogenization or deletion. st6gal2 and st6gal2-r were maintained on DRE 6 and DRE 9. D, conserved synteny observed between the amphioxus genomic region hosting the st6gal-like gene (scaf V2 104q) and the two sets of human paralogons described in the legend to Fig. 3.

Article Snippet: In vertebrates, most examined genomes contain two members the st6gal1 and st6gal2 paralogous genes, except in the lamprey Petromyzon marinus , where three st6gal copies were found.

Techniques: Derivative Assay, Blocking Assay, Translocation Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular Phylogeny and Functional Genomics of ?-Galactoside ?2,6-Sialyltransferases That Explain Ubiquitous Expression of st6gal1 Gene in Amniotes *

doi: 10.1074/jbc.M110.163931

Figure Lengend Snippet: Expression pattern of the zebrafish and amphibian st6gal genes in various adult tissues using RT-PCR. A, fish st6gal genes. Relative expression levels of zebrafish st6gal and β-actin mRNA were evaluated by RT-PCR as described under “Experimental Procedures,” among various adult tissues. Oligonucleotide primer sequences specific to fish st6gal1, st6gal2, and st6gal2-r are given in supplemental Fig. 5 and yield 896-, 628-, and 299-bp PCR fragments, respectively. Lane 1, kidney; lane 2, intestine; lane 3, brain; lane 4, liver; lane 5, muscle; lane 6, heart; lane 7, eggs; lane 8, oocytes. The zebrafish β-actin (378 bp) was amplified as a control of cDNA synthesis and purity. B, amphibian st6gal genes. Oligonucleotides specific to S. tropicalis st6gal1 and st6gal2 are indicated in supplemental Fig. 5 and yield 236- and 465-bp PCR fragments, respectively. Lane 1, muscle; lane 2, eyes; lane 3, heart; lane 4, skin; lane 5, lung; lane 6, stomach; lane 7, ovaries; lane 8, intestine; lane 9, liver; lane 10, brain; lane 11, kidney. The amphibian β-catenin (327 bp) was amplified for 35 cycles as a control of cDNA synthesis and purity.

Article Snippet: In vertebrates, most examined genomes contain two members the st6gal1 and st6gal2 paralogous genes, except in the lamprey Petromyzon marinus , where three st6gal copies were found.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Control, cDNA Synthesis

Journal: The Journal of Biological Chemistry

Article Title: Molecular Phylogeny and Functional Genomics of ?-Galactoside ?2,6-Sialyltransferases That Explain Ubiquitous Expression of st6gal1 Gene in Amniotes *

doi: 10.1074/jbc.M110.163931

Figure Lengend Snippet: Expression pattern of the zebrafish st6gal genes during zebrafish embryonic development using whole mount ISH at early developmental stages (A) and later developmental stages (B). The st6gal1 gene shows no labeling at early developmental stages (gastrula (G), early somitogenesis (ES), and middle somitogenesis (MS)). st6gal1 is found in the ventricular part of telencephalon, in the ventral part of diencephalon, in the lateral part of the hindbrain (discontinuous patches of cells), and in the lateral part of the spinal cord at 24 and 36 hpf. st6gal1 gene expression is detected in the ganglion cell layer of the retina (except in the proliferative zone), cranial ganglia, and brain marginal zone at 48 hpf and is not detected later. The st6gal2 gene is expressed at the gastrula stage in the prechordal plate mesendoderm and at early somitogenesis in polster, anterior border of neural plate, adaxial cells, formed somites, and also weakly in segmental plates. At middle somitogenesis, it is expressed in polster, anterior head epidermis, caudal adaxial cells, and the last formed somites. At 24 hpf, the st6gal2 gene is expressed in tail somites and adaxial cells and weakly in the CNS; later, at 36 hpf, a stronger signal is found in the marginal zone, and no labeling is detected in tectum. At 48 hpf and 5 days postfertilization, the st6gal2 transcript is detected in anterior diencephalon and in the ganglion cell layer of retina but not in the proliferative zone. The st6gal2-r gene is weakly expressed at the gastrula stage and shows also a weak basal level of expression at early somitogenesis and middle somitogenesis and more labeling in hatching glands. At later developmental stages, from pharyngula to larva stages (24 hpf to 5 days postfertilization (dpf)), the st6gal2-r gene is detected in hatching glands, mucus cells, proctodeum, intestinal bulb, gall bladder, inner nuclear cell layer of retina (except in the proliferative zone), nucleus in diencephalon, nucleus in anterior tectum, and nucleus in hindbrain at the larval stage. hg, hatching gland; inl of retina, inner nuclear layer of retina; i bulb, intestinal bulb; n in diencephalon, nucleus in diencephalon; n in ant tectum, nucleus in anterior tectum; n in hb, nucleus in hindbrain; up mesoderm, unsegmented paraxial mesoderm; tg, tegmentum; ventral dc, ventral diencephalon. LV, lateral view; DV, dorsal view.

Article Snippet: In vertebrates, most examined genomes contain two members the st6gal1 and st6gal2 paralogous genes, except in the lamprey Petromyzon marinus , where three st6gal copies were found.

Techniques: Expressing, Labeling, Gene Expression