Abstract:Proper N-glycosylation of proteins is important for normal brain development and nervous system function. Identification of the localization, carrier proteins and interacting partners of N-glycans is essential for understanding the roles of glycoproteins. The present study examined the N-glycan A2G´2F (Galβ1-3GlcNAcβ1-A2G´2F has a branched sialic acid structural feature, and branched sialylated A2G´2F is a major N-glycan in the mouse brain. Its expression in the mouse brain increases during development, sugges… Show more
“…in developing mouse brains and may mediate interactions between dendritic spines and microglia (42). Although glycoproteins and their binding partners still need to be identified in echinoderms, it is known that their neuronal systems display similarities to those in chordates (43).…”
Echinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea. To this end, we performed a multi-step N-glycan analysis by HPLC and various exoglyosidase and chemical treatments in combination with MALDI-TOF MS and MS/MS. Using this approach, we found a wealth of hybrid and complex oligosaccharide structures reminiscent of those in higher vertebrates as well as some classical invertebrate glycan structures. 70% of these N-glycans were anionic, carrying either sialic acid, sulfate, or phosphate residues. In terms of glycophylogeny, our data position the brittle star between invertebrates and vertebrates and confirm the high diversity of N-glycosylation in lower organisms. Figure 4. RP HPLC fractionation of anionic N-glycans. A-K, for the first dimension, anionic enriched N-glycans were separated on a Kinetex C18 RP HPLC (pH 6; calibrated in terms of glucose units). The color code and letters indicate pooled peaks whose second-dimension HIAX-HPLC profiles are shown in Fig. S5, A-K. Fractions are annotated with example glycans according to the symbol nomenclature for glycans (where Me and S represent methyl and sulfate, respectively). The illustrated structures are based on a combination of MS/MS analysis, RP and HIAX-HPLC elution times, as well as enzymatic and/or chemical treatments. Because of the separation properties of the RP column used as the first dimension, multiple sulfated structures are shown in A, modified hybrid structures in B, multiple sialylated biantennary structures in C-E, and those with methylated N-glycolylneuraminic acid and multiantennary structures in G-K. The 4 g.u. RP HPLC peak not subject to 2D-HPLC contains a nondigestible glycan not related to other analyzed structures.
Brittle star N-glycome
“…in developing mouse brains and may mediate interactions between dendritic spines and microglia (42). Although glycoproteins and their binding partners still need to be identified in echinoderms, it is known that their neuronal systems display similarities to those in chordates (43).…”
Echinoderms are among the most primitive deuterostomes and have been used as model organisms to understand chordate biology because of their close evolutionary relationship to this phylogenetic group. However, there are almost no data available regarding the N-glycomic capacity of echinoderms, which are otherwise known to produce a diverse set of species-specific glycoconjugates, including ones heavily modified by fucose, sulfate, and sialic acid residues. To increase the knowledge of diversity of carbohydrate structures within this phylum, here we conducted an in-depth analysis of N-glycans from a brittle star (Ophiactis savignyi) as an example member of the class Ophiuroidea. To this end, we performed a multi-step N-glycan analysis by HPLC and various exoglyosidase and chemical treatments in combination with MALDI-TOF MS and MS/MS. Using this approach, we found a wealth of hybrid and complex oligosaccharide structures reminiscent of those in higher vertebrates as well as some classical invertebrate glycan structures. 70% of these N-glycans were anionic, carrying either sialic acid, sulfate, or phosphate residues. In terms of glycophylogeny, our data position the brittle star between invertebrates and vertebrates and confirm the high diversity of N-glycosylation in lower organisms. Figure 4. RP HPLC fractionation of anionic N-glycans. A-K, for the first dimension, anionic enriched N-glycans were separated on a Kinetex C18 RP HPLC (pH 6; calibrated in terms of glucose units). The color code and letters indicate pooled peaks whose second-dimension HIAX-HPLC profiles are shown in Fig. S5, A-K. Fractions are annotated with example glycans according to the symbol nomenclature for glycans (where Me and S represent methyl and sulfate, respectively). The illustrated structures are based on a combination of MS/MS analysis, RP and HIAX-HPLC elution times, as well as enzymatic and/or chemical treatments. Because of the separation properties of the RP column used as the first dimension, multiple sulfated structures are shown in A, modified hybrid structures in B, multiple sialylated biantennary structures in C-E, and those with methylated N-glycolylneuraminic acid and multiantennary structures in G-K. The 4 g.u. RP HPLC peak not subject to 2D-HPLC contains a nondigestible glycan not related to other analyzed structures.
Brittle star N-glycome
“…Although the first Ig-domain of Siglec-H carries all the characteristic features for sialic acid binding, no binding to sialic acids had been detected so far by various attempts ( 22 ). Recently, Siglec-H binding to branched sialylated N-glycans isolated from mouse brain was demonstrated ( 52 ), which may be relevant for Siglec-H on microglia, the significance of this finding for mouse pDCs is unclear. Siglec-H does not have a clear orthologue in the human, but potential functional homologues with similar structures are Siglec-14 or Siglec-16 ( 19 ).…”
Siglec-H is a DAP12-associated receptor on plasmacytoid dendritic cells (pDCs) and microglia. Siglec-H inhibits TLR9-induced IFN-α production by pDCs. Previously, it was found that Siglec-H-deficient mice develop a lupus-like severe autoimmune disease after persistent murine cytomegalovirus (mCMV) infection. This was due to enhanced type I interferon responses, including IFN-α. Here we examined, whether other virus infections can also induce autoimmunity in Siglec-H-deficient mice. To this end we infected Siglec-H-deficient mice with influenza virus or with Lymphocytic Choriomeningitis virus (LCMV) clone 13. With both types of viruses we did not observe induction of autoimmune disease in Siglec-H-deficient mice. This can be explained by the fact that both types of viruses are ssRNA viruses that engage TLR7, rather than TLR9. Also, Influenza causes an acute infection that is rapidly cleared and the chronicity of LCMV clone 13 may not be sufficient and may rather suppress pDC functions. Siglec-H inhibited exclusively TLR-9 driven type I interferon responses, but did not affect type II or type III interferon production by pDCs. Siglec-H-deficient pDCs showed impaired Hck expression, which is a Src-family kinase expressed in myeloid cells, and downmodulation of the chemokine receptor CCR9, that has important functions for pDCs. Accordingly, Siglec-H-deficient pDCs showed impaired migration towards the CCR9 ligand CCL25. Furthermore, autoimmune-related genes such as Klk1 and DNase1l3 are downregulated in Siglec-H-deficient pDCs as well. From these findings we conclude that Siglec-H controls TLR-9-dependent, but not TLR-7 dependent inflammatory responses after virus infections and regulates chemokine responsiveness of pDCs.
“…As one of the most common, significant, and complex PTM occurring after protein translation [ 55 ], N-glycosylation plays a crucial role in protein biogenesis and function. Besides its potential involvement in regulating brain development and function [ 56 ], N-glycosylation exerts its important effects by influencing protein folding, cellular localization, stability, and solubility. Fig.…”
Section: Characteristics and Functions Of Glycosylationmentioning
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