N-glycan structures of β-HlH subunit of Helix lucorum hemocyanin (original) (raw)
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Molluscan hemocyanins (Hcs) have recently particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. The oligosaccharide structures of the structural subunits RvH2 from Rapana venosa hemocyanin (RvH) and βc-Helix lucorum hemocyanin (βc-HlH) were comparative investigated by mass spectrometry. Our study revealed a highly heterogeneous mixture of N-glycans with compositions Hex 3-7 HexNAc 2-5 MeHex 0-4 Pent 0-1 Fuc 0-1 and Hex 0-9 HexNAc 2-4 MeHex 0-1 HexA 0-1 Pent 0-1 Fuc 0-3 , isolation from βc-HlH and RvH2, respectively. N-glycans from βc-HlH contain mainly a terminal methyl-hexose (MeHex) residue, in some cases even more than one. Several carbohydrate moieties from βc-HlH are core-fucosylated, and also possess on methylation. In contrast with RvH2, N-glycans with an internal fucose residue, substituted at two positions with N-acetylhexosamine and hexuronic acid, as and glycans containing internal fucose were not found in βc-HlH. The glycosylation sites occupancy was subsequently elucidated by precursor ion scanning of the intact glycopeptides from RvH and βc-HlH using a nano-ESI mass spectrometry and Q-trap-LC/MS system. The oligosaccharide moieties found in HlH and RvH reveal a complex N-glycan pattern combining typical structural features of different higher organisms. Moreover, they are a potential source of novel N-glycans that are important for the stimulation of the immune response and/or for the production of antibodies used in diagnosis and therapy.
Mass spectral evidence for N-glycans with branching on fucose in a molluscan hemocyanin
Biochemical and Biophysical Research Communications, 2005
Glycopeptides, isolated from a trypsinolysate of functional unit (FU) RtH2-e of Rapana thomasiana hemocyanin subunit 2, were analysed by electrospray ionization mass spectrometry and MS/MS. From the molecular mass observed after deglycosylation, it was inferred that all glycopeptides shared the same peptide stretch 92-143 of FU RtH2-e with a glycosylation site at Asn-127. Besides the core structure Man 3 GlcNAc 2 for N-glycosylation, structures with a supplementary GlcNAc linked to either the Man(a1-3) or the Man(a1-6) arm and/or an additional tetrasaccharide unit connected to the other Man arm were observed, indicating the existence of microheterogeneity at the glycan level. The tetrasaccharide unit contains a central fucose moiety substituted with 3-O-methylgalactose and N-acetylgalactosamine, and linked to GlcNAc at the reducing end. This structure represents a novel N-glycan motif and is likely to be immunogenic. A second potential site for N-glycosylation in FU RtH2-e at Asn-17 was shown to be not glycosylated.
De Novo Structural Determination of the Oligosaccharide Structure of Hemocyanins from Molluscs
Biomolecules
A number of studies have shown that glycosylation of proteins plays diverse functions in the lives of organisms, has crucial biological and physiological roles in pathogen–host interactions, and is involved in a large number of biological events in the immune system, and in virus and bacteria recognition. The large amount of scientific interest in glycoproteins of molluscan hemocyanins is due not only to their complex quaternary structures, but also to the great diversity of their oligosaccharide structures with a high carbohydrate content (2–9%). This great variety is due to their specific monosaccharide composition and different side chain composition. The determination of glycans and glycopeptides was performed with the most commonly used methods for the analysis of biomolecules, including peptides and proteins, including Matrix Assisted Laser Desorption/Ionisation–Time of Flight (MALDI-TOF-TOF), Liquid Chromatography - Electrospray Ionization-Mass Spectrometry (LC/ESI-MS), Liqui...
European Journal of Biochemistry, 1997
The primary structures of 21 novel monoantennary and diantennary N-glycans of the glycoprotein a,-hemocyanin (a,-Hc) of Helix pnmutia have been determined. Outer oligosaccharide fragments (antennae) were released from the glycoprotein by Smith degradation of an a,-Hc pronase digest. The major antenna, obtained following HPLC fractionation on Lichrosorb-NH,, was characterized using 'H-NMR spectroscopy, fast-atom-bombardment mass spectrometry, and linkage analysis, and corresponds to a pentasaccharide fragment. The intact carbohydrate chains of a,,-Hc were released with peptide-N4-(Nacetyl-P-glucosaminy1)asparagine amidase-F digestion, separated from the protein on Bio-Gel P-100, and subfractionated on Bio-Gel P-4. A portion of subfractions was reduced with sodium borodeuteride, and the non-reduced and reduced samples were further fractionated on CarboPac PA-1, Lichrosorb-NH,/ Lichrosphere-NH2, and/or Lichrosphere-C,,. Purified oligosaccharides and oligosaccharide-alditols were analyzed using 500/600-MHz 'H-NMR spectroscopy. In total, four novel types of antenna were identified, namely,
European Journal of Biochemistry, 2002
Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N-linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two-dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI-TOF-MS, ESI-ion trap-MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose-type glycans and truncated sugar chains derived thereof. As a characteristic feature, a number of the studied N-glycans contained a Gal(b1-6)Man-unit which has not been found in glycoprotein-N-glycans so far. Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements.
Positions of the Glycans in Molluscan Hemocyanin, Determined by Fluorescence Spectroscopy
Journal of Fluorescence, 2013
Molluscan hemocyanins are glycoproteins with different quaternary and carbohydrate structures. It was suggested that the carbohydrate chains of some Hcs are involved in their antiviral and antitumor effect, as well in the organization of the quaternary structure of the molecules. Using a well-known complex for saccharide sensing, positions and access to the carbohydrate chains in the native hemocyanins from Rapana venosa (RvH) and Helix lucorum (HlH) and also their structural subunits (RvH1, RvH2 and β c HlH) and functional units (FUs) were analysed by fluorescence spectroscopy and circular dichroism. Almost no effect was observed in the fluorescence emission after titration of the complex with native RvH and HlH due to lack of free hydroxyl groups which are buried in the didecameric form of the molecules. Titration with the structural subunits β c HlH and RvH2, increasing of the emission indicates the presence of free hydroxyl groups compared to the native molecules. Complex titration with the structural subunit βc-HlH of H. lucorum Hcs leads to a 2.5 fold increase in fluorescence intensity. However, the highest emission was measured after titration of the complex with FU βcHlH-g. The result was explained by the structural model of β c HlH-g showing the putative position of the glycans on the surface of the molecule. The results of the fluorescent measurements are in good correlation with those of the circular dichroism data, applied to analyse the effect of titration on the secondary structure of the native molecules and functional units. The results also support our previously made suggestion that the N-linked oligosaccharide trees are involved in the quaternary organization of molluscan Hcs.
Journal of Biological Chemistry, 2019
Hemocyanins are widely used as carriers, adjuvants, and nonspecific immunostimulants in cancer because they promote Th1 immunity in mammals. Hemocyanins also interact with glycan-recognizing innate immune receptors on antigen-presenting cells, such as the C-type lectin immune receptors MR and MGL and the Toll-like receptors (TLRs), stimulating proinflammatory cytokine secretion. However, the role of N-linked oligosaccharides on the structural and immunological properties of hemocyanin is unclear. Mollusk hemocyanins, such as CCH (Concholepas concholepas), FLH (Fissurella latimarginata) and KLH (Megathura crenulata), are oligomeric glycoproteins with complex dodecameric quaternary structures and heterogeneous glycosylation patterns, primarily consisting of mannose-rich N-glycans. Here, we report that enzyme-catalyzed N-deglycosylation of CCH, FLH and KLH disrupts their quaternary
Proceedings of the Bulgarian Academy of Sciences
Glycosylation plays an important role in many important processes in organisms. Their participation in the construction of the tertiary structure and immunotherapeutic activity of the hemocyanin Rapana venosa (RvH) from the Black Sea has been presented. Based on these results, the importance of glycans in organization of the structure of hemocyanin from the abdominal mollusk Haliotis tuberculata (HtH) was also studied. After incubation of both isoforms HtH1 and HtH2 with yeast Saccharomyces cerevisiae and enzyme Zymolyase, fractions with lower molecular weight (MW) between 45 and 65 kDa, which corresponds to MW of functional units (FUs) of hemocyanins, was identified. A comparative analysis of 10% SDS PAGE assays confirmed the depolymerization of the subunits HtH1 and HtH2 due to the specific glycosidase in the yeast S. cerevisiae and the enzyme Zymolyase. The presented results confirm our hypothesis that glycans are involved in the formation of the structural subunits of HtH.
Glycan structures of the structural subunit (HtH1) of Haliotis tuberculata hemocyanin
Glycoconjugate Journal, 2011
Hemocyanins are giant extracellular oxygen carriers in the hemolymph of many molluscs and arthropods with different quaternary structure. They are represented in the hemolymph of molluscs with one, two or three isoforms, as decameric, didecameric, multidecameric and tubules aggregates. We describe here the structure of the hemocyanin Helix lucorum (HlH), species in the series of molluscan hemocyanins. In contrast with other molluscan hemocyanins, three different hemocyanin isopolypeptides were isolated from the hemolymph of the garden snail H. lucorum, named as β-HlH, α D -HlH and α N -HlH. Their molecular masses were determined by size exclusion chromatography to be 1068 kDa (β-HlH) and 1079 kDa (α D -HlH, and α N -HlH). Native HlH exhibits a predominant didecameric structure as revealed by electron microscopy and additionally few tridecamers are shown in the electron micrographs of HlH resulting from the association of a further decamer with one didecamer. The three isoforms are represented mainly as homogeneous didecamers, but they have different behaviour after dissociation and reassociation in the pH-stabilizing buffer, containing 20 mM CaCl 2 . All isoforms were reassociated into didecamers and tubules with different length, but in contrast to α D -HlH isoform, longer tubules were observed in β-HlH. Moreover the structure of β-HlH was analysed after limited proteolysis with trypsin followed by FPLC and HPLC separation of the cleavage products. Eight different functional units were identified by their N-terminal sequences and molecular masses. The protein characteristics, including UV absorption at 340 nm, fluorescence and CD spectra of the native molecule and its units confirmed the structure of multimer protein complexes.