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- Persson, Andrea, et al.
(författare)
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LC-MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 293:26, s. 10202-10219
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Tidskriftsartikel (refereegranskat)abstract
- Structural characterization of glycosaminoglycans remains a challenge but is essential for determining structure-function relationships between glycosaminoglycans and the biomolecules with which they interact and for gaining insight into the biosynthesis of glycosaminoglycans. We have recently reported that xyloside-primed chondroitin/dermatan sulfate derived from a human breast carcinoma cell line, HCC70, has cytotoxic effects and shown that it differs in disaccharide composition from nontoxic chondroitin/dermatan sulfate derived from a human breast fibroblast cell line, CCD-1095Sk. To further investigate the structural requirements for the cytotoxic effect, we developed a novel LC-MS/MS approach based on reversed-phase dibutylamine ion-pairing chromatography and negative-mode higher-energy collision dissociation and used it in combination with cell growth studies and disaccharide fingerprinting. This strategy enabled detailed structural characterization of linkage regions, internal oligosaccharides, and nonreducing ends, revealing not only differences between xyloside-primed chondroitin/dermatan sulfate from HCC70 cells and CCD-1095Sk cells, but also sialylation of the linkage region and previously undescribed methylation and sulfation of the nonreducing ends. Although the xyloside-primed chondroitin/dermatan sulfate from HCC70 cells was less complex in terms of presence and distribution of iduronic acid than that from CCD-1095Sk cells, both glucuronic acid and iduronic acid appeared to be essential for the cytotoxic effect. Our data have moved us one step closer to understanding the structure of the cytotoxic chondroitin/dermatan sulfate from HCC70 cells primed on xylosides and demonstrate the suitability of the LC-MS/MS approach for structural characterization of glycosaminoglycans.
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| 2. |
- Stenfelt, Linn, et al.
(författare)
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Missense mutations in the C-terminal portion of the B4GALNT2-encoded glycosyltransferase underlying the Sd(a−) phenotype
- 2019
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Ingår i: Biochemistry and Biophysics Reports. - : Elsevier BV. - 2405-5808. ; 19
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Tidskriftsartikel (refereegranskat)abstract
- Sda is a high-frequency carbohydrate histo-blood group antigen, GalNAcβ1-4(NeuAcα2-3)Galβ, implicated in pathogen invasion, cancer, xenotransplantation and transfusion medicine. Complete lack of this glycan epitope results in the Sd(a−) phenotype observed in 4% of individuals who may produce anti-Sda. A candidate gene (B4GALNT2), encoding a Sda-synthesizing β-1,4-N-acetylgalactosaminyltransferase (β4GalNAc-T2), was cloned in 2003 but the genetic basis of human Sda deficiency was never elucidated. Experimental and bioinformatic approaches were used to identify and characterize B4GALNT2 variants in nine Sd(a−) individuals. Homozygosity for rs7224888:T > C dominated the cohort (n = 6) and causes p.Cys466Arg, which targets a highly conserved residue located in the enzymatically active domain and is judged deleterious to β4GalNAc-T2. Its allele frequency was 0.10–0.12 in different cohorts. A Sd(a−) compound heterozygote combined rs7224888:T > C with a splice-site mutation, rs72835417:G > A, predicted to alter splicing and occurred at a frequency of 0.11–0.12. Another compound heterozygote had two rare nonsynonymous variants, rs148441237:A > G (p.Gln436Arg) and rs61743617:C > T (p.Arg523Trp), in trans. One sample displayed no differences compared to Sd(a+). When investigating linkage disequilibrium between B4GALNT2 variants, we noted a 32-kb block spanning intron 9 to the intergenic region downstream of B4GALNT2. This block includes RP11-708H21.4, a long non-coding RNA recently reported to promote tumorigenesis and poor prognosis in colon cancer. The expression patterns of B4GALNT2 and RP11-708H21.4 correlated extremely well in >1000 cancer cell lines. In summary, we identified a connection between variants of the cancer-associated B4GALNT2 gene and Sda, thereby establishing a new blood group system and opening up for the possibility to predict Sd(a+) and Sd(a‒) phenotypes by genotyping. © 2019 The Authors
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