Glycosylated proteins preserved over millennia: N-glycan analysis of Tyrolean Iceman, Scythian Princess and Warrior
Süreyya ÖzcanBum Jin KimGrace S. RoJae‐Han KimThomas BereuterChristian ReiterLauren M. DimapasocDaniel GarridoDavid A. MillsRudolf GrimmCarlito B. LebrillaHyun Joo An
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An improved understanding of glycosylation will provide new insights into many biological processes. In the analysis of oligosaccharides from biological samples, a strict regime is typically followed to ensure sample integrity. However, the fate of glycans that have been exposed to environmental conditions over millennia has not yet been investigated. This is also true for understanding the evolution of the glycosylation machinery in humans as well as in any other biological systems. In this study, we examined the glycosylation of tissue samples derived from four mummies which have been naturally preserved: – the 5,300 year old “Iceman called Oetzi”, found in the Tyrolean Alps; the 2,400 year old “Scythian warrior” and “Scythian Princess”, found in the Altai Mountains; and a 4 year old apartment mummy, found in Vienna/Austria. The number of N-glycans that were identified varied both with the age and the preservation status of the mummies. More glycan structures were discovered in the contemporary sample, as expected, however it is significant that glycan still exists in the ancient tissue samples. This discovery clearly shows that glycans persist for thousands of years and these samples provide a vital insight into ancient glycosylation, offering us a window into the distant past.In a soil bioassay, adult Deroceras reticulatum (Stylommatophora: Limacidae) and three different weight-classes of young Arion lusitanicus (Stylommatophora: Arionidae) were exposed to a single dosage (170 dauer larvae per g of soil) of the nematode Phasmarhabditis hermaphrodita monoxenically associated with the bacterium Moraxella osloensis. Groups of 10 slugs were continuously exposed to nematodes for 4 days, and then transferred individually to Petri-dishes containing a disc of Chinese cabbage as food. Food consumption—measured by image analysis—and slug mortality were recorded daily for 10 days. Food consumption was inhibited in both slug species tested. D. reticulatum stopped feeding 6 days after the start of nematode treatment, while all A. lusitanicus continued to feed. However, in the three weight-classes of A. lusitanicus (0.15 g, 0.24 g, 0.45 g), food consumption was reduced by at least 50 %. The greatest reduction in feeding, nearly 90 %, was noted in the smallest A. lusitanicus. The nematodes successfully killed D. reticulatum but were less efficient at killing young A. lusitanicus. At the end of the experiment, mortality was highest in D. reticultatum (98 %) and the smallest weight-class of A. lusitanicus (47 %). There was almost no mortality in the largest weight-class of A. lusitanicus treated with nematodes. P. hermaphrodita associated with M. osloensis can thus be considered as a biological control agent for young stages of A. lusitanicus for its effect as a feeding inhibitor, rather than for its ability to kill the slugs.
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Recombinant proteins represent almost half of the top selling therapeutics-with over a hundred billion dollars in global sales-and their efficacy and safety strongly depend on glycosylation. In this study, we showcase a simple method to simultaneously analyze N-glycan micro- and macroheterogeneity of an immunoglobulin G (IgG) by quantifying glycan occupancy and distribution. Our approach is linear over a wide range of glycan and glycoprotein concentrations down to 25 ng/mL. Additionally, we present a case study demonstrating the effect of small molecule metabolic regulators on glycan heterogeneity using this approach. In particular, sodium oxamate (SOD) decreased Chinese hamster ovary (CHO) glucose metabolism and reduced IgG glycosylation by 40% through upregulating reactive oxygen species (ROS) and reducing the UDP-GlcNAc pool, while maintaining a similar glycan profile to control cultures. Here, we suggest glycan macroheterogeneity as an attribute should be included in bioprocess screening to identify process parameters that optimize culture performance without compromising antibody quality.
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In response to DNA damage, p53 undergoes post-translational modifications (including acetylation) that are critical for its transcriptional activity. However, the mechanism by which p53 acetylation is regulated is still unclear. Here, we describe an essential role for HLA-B-associated transcript 3 (Bat3)/Scythe in controlling the acetylation of p53 required for DNA damage responses. Depletion of Bat3 from human and mouse cells markedly impairs p53-mediated transactivation of its target genes Puma and p21 . Although DNA damage-induced phosphorylation, stabilization, and nuclear accumulation of p53 are not significantly affected by Bat3 depletion, p53 acetylation is almost completely abolished. Bat3 forms a complex with p300, and an increased amount of Bat3 enhances the recruitment of p53 to p300 and facilitates subsequent p53 acetylation. In contrast, Bat3-depleted cells show reduced p53–p300 complex formation and decreased p53 acetylation. Furthermore, consistent with our in vitro findings, thymocytes from Bat3-deficient mice exhibit reduced induction of puma and p21, and are resistant to DNA damage-induced apoptosis in vivo. Our data indicate that Bat3 is a novel and essential regulator of p53-mediated responses to genotoxic stress, and that Bat3 controls DNA damage-induced acetylation of p53.
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Glycosylation changes are often described in different physiological processes and pathological conditions and as a lifestyle effect. Alterations in cancer cells metabolism result in the production of altered glycan structures, which are being recognized by the immune system that result in generation of novel anti-glycan antibodies. Presence and screening of antibodies was performed using glycan arrays. Immunoglobulin G (IgG) glycosylation was performed using plasma purification and glycan chromatography. Results show significant differences between healthy individuals and those with cancer. Antibody binding to 24/48 glycans give positive results with great statistical significance between studied sample groups. IgG glycosylation analysis shows that 10/23 glycan chromatographic peaks are changed in colorectal cancer and that galactosylation is one of the main indicators of appearance and progression of the disease. Understanding of IgG glycosylation changes and protein-glycan interactions, and the fact they are effective in numerous diseases are important for understanding biology of cancer cells.
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Abstract Human genetic diseases that affect N‐glycosylation result from the defective synthesis of the N‐linked sugar moiety (glycan) of glycoproteins. The role of glycans for proper protein folding and biological functions is illustrated in the variety and severity of clinical manifestations shared by congenital disorders of glycosylation (CDG). This family of inherited metabolic disorders includes defects in the assembly of the oligosaccharide precursor that lead to an under‐occupancy of N‐glycosylation sites (CDG‐I), and defects of glycan remodeling (CDG‐II). Mass spectrometry constitutes a key tool for characterization of CDG‐I defects by mass resolution of native protein glycoforms that differ for glycosylation‐site occupancy. Glycan MS analyses in CDG‐II is mandatory to detect whenever possible a repertoire of structures to pinpoint candidate enzymes and genes responsible for the abnormal N‐glycan synthesis. In this manuscript, we review the MS applications in the area of CDG and related disorders with a special emphasis on those techniques that have been already applied or might become functional for diagnosis, characterization, and treatment monitoring in some specific conditions. © 2008 Wiley Periodicals, Inc., Mass Spec Rev 28:517–542, 2009
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HLA-B-associated transcript 3 (BAT3) was originally identified as one of the genes located within human major histocompatibility complex. It encodes a large proline-rich protein with unknown function. In this study, we found that a fragment of the BAT3 gene product interacts with a candidate tumor suppressor, DAN, in the yeast-based two-hybrid system. We cloned the full-length rat BAT3 cDNA from a fibroblast 3Y1 cDNA library. Our sequence analysis has demonstrated that rat BAT3 cDNA is 3617 nucleotides in length and encodes a full-length BAT3 (1098 amino acids) with an estimated molecular mass of 114,801 daltons, which displays an 87.4% identity with human BAT3. The deletion experiment revealed that the N-terminal region (amino acid residues 1-80) of DAN was required for the interaction with BAT3. Green fluorescent protein-tagged BAT3 was largely localized in the cytoplasm of COS cells. Northern hybridization showed that BAT3 mRNA was expressed in all the adult rat tissues examined but predominantly in testis. In addition, the level of BAT3 mRNA expression was more downregulated in some of the transformed cells, including v-mos- and v-Ha-ras-transformed 3Y1 cells, than in the parental cells.
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This chapter contains sections titled: Introduction – Glycosylated Proteins Basic Building Blocks of Glycosylation in Human Cells Formation of Complex Glycan Structures Glycan Synthesis is Catalyzed by Enzymes of Glycosylation – the "Glycozymes" Protein Glycosylation – Relationship Between N-linked and O-linked Glycoproteins N-linked Glycoproteins O-linked Glycoproteins O- and N-linked Glycan Chain Extension and Commonly Occurring Glycan Motifs Analytical Methodologies Developed to Detect and Characterize Glycosylation Conclusion References
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