ABSTRACT
A major puzzle is: are all glycoproteins routed through the ER calnexin pathway irrespective of whether this is required for their correct folding? Calnexin recognizes the terminal Glcalpha1-3Manalpha linkage, formed by trimming of the Glcalpha1-2Glcalpha1-3Glcalpha1-3Manalpha (Glc3Man) unit in Glc3Man9GlcNAc2. Different conformations of this unit have been reported. We have addressed this problem by studying the conformation of a series of N-glycans; i.e. Glc3ManOMe, Glc3Man(4,5,7)GlcNAc2 and Glc1Man9GlcNAc2 using 2D NMR NOESY, ROESY, T-ROESY and residual dipolar coupling experiments in a range of solvents, along with solution molecular dynamics simulations of Glc3ManOMe. Our results show a single conformation for the Glcalpha1-2Glcalpha and Glcalpha1-3Glcalpha linkages, and a major (65%) and a minor (30%) conformer for the Glcalpha1-3Manalpha linkage. Modeling of the binding of Glc1Man9GlcNAc2 to calnexin suggests that it is the minor conformer that is recognized by calnexin. This may be one of the mechanisms for controlling the rate of recruitment of proteins into the calnexin/calreticulin chaperone system and enabling proteins that do not require such assistance for folding to bypass the system. This is the first time evidence has been presented on glycoprotein folding that suggests the process may be optimized to balance the chaperone-assisted and chaperone-independent pathways.
Subject(s)
Glycoproteins/chemistry , Glycoproteins/metabolism , Molecular Chaperones/metabolism , Oligosaccharides/chemistry , Protein Folding , Acetylglucosamine/chemistry , Animals , CHO Cells , Calnexin/metabolism , Carbohydrate Conformation , Carbohydrate Sequence , Chickens , Computer Simulation , Cricetinae , Cricetulus , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Protons , Solubility , Solvents , Time Factors , WaterABSTRACT
The mannose receptor (MR) is a heavily glycosylated endocytic receptor that recognizes both mannosylated and sulfated ligands through its C-type lectin domains and cysteine-rich (CR) domain, respectively. Differential binding properties have been described for MR isolated from different sources, and we hypothesized that this could be due to altered glycosylation. Using MR transductants and purified MR, we demonstrate that glycosylation differentially affects both MR lectin activities. MR transductants generated in glycosylation mutant cell lines lacked most mannose internalization activity, but could internalize sulfated glycans. Accordingly, purified MR bearing truncated Man5-GlcNAc2 glycans (Man5 -MR) or non-sialylated complex glycans (SA0-MR) did not bind mannosylated glycans, but could recognize SO4-3-Gal in vitro. Additional studies showed that, although mannose recognition was largely independent of the oligomerization state of the protein, recognition of sulfated carbohydrates was mostly mediated by self-associated MR and that, in SA0-MR, there was a higher proportion of oligomeric MR. These results suggest that self-association could lead to multiple presentation of CR domains and enhanced avidity for sulfated sugars and that non-sialylated MR is predisposed to oligomerize. Therefore, the glycosylation of MR, terminal sialylation in particular, could influence its binding properties at two levels. (i) It is required for mannose recognition; and (ii) it modulates the tendency of MR to self-associate, effectively regulating the avidity of the CR domain for sulfated sugar ligands.
Subject(s)
Lectins, C-Type/chemistry , Lectins/chemistry , Macrophages/metabolism , Mannose-Binding Lectins/chemistry , Receptors, Cell Surface/chemistry , Animals , Blotting, Western , CHO Cells , Carbohydrates/chemistry , Cell Membrane/metabolism , Cell Separation , Chromatography, Gel , Cricetinae , Endocytosis , Flow Cytometry , Glycosylation , Ligands , Mannose/chemistry , Mannose Receptor , Mice , Microscopy, Confocal , Models, Molecular , Mutation , Polysaccharides/chemistry , Protein Binding , Protein Structure, Tertiary , Retroviridae/genetics , Sulfur/chemistry , Surface Plasmon Resonance , Time Factors , Tissue DistributionSubject(s)
Lectins, C-Type/metabolism , Ligands , Mannose-Binding Lectins/metabolism , Receptors, Cell Surface/metabolism , Acetylglucosamine/chemistry , Animals , CHO Cells , Cricetinae , Glycosylation , Lectins, C-Type/chemistry , Mannose/chemistry , Mannose/metabolism , Mannose Receptor , Mannose-Binding Lectins/chemistry , Protein Binding , Receptors, Cell Surface/chemistryABSTRACT
Glycosylation is of importance for the structure and function of proteins. In the case of vitellin (Vt), a ubiquitous protein accumulated into granules as the main yolk protein constituent of oocytes during oogenesis, glycosylation could be of importantance for the folding, processing and transport of the protein to the yolk and also provides a source of carbohydrate during embryogenesis. Vt from the crayfish Cherax quadricarinatus is synthesized as a precursor protein, vitellogenin (Vg), in the hepatopancreas, transferred to the hemolymph, and mobilized into the growing oocyte via receptor-mediated endocytosis. The gene sequence of C. quadricarinatus shows a 2584-amino-acid protein with 10 putative glycosylation sites. In this study a combined approach of lectin immunoblotting, in-gel deglycosylation, and mass spectrometry was used to identify the glycosylation sites and probe the structure of the glycan moieties using C. quadricarinatus Vg as a model system. Three of the consensus sites for N-glycosylation-namely, Asn(152), Asn(160) and Asn(2493)-were glycosylated with the high-mannose glycans, Man(5-9)GlcNAc(2), and the glucose-capped oligosaccharide Glc(1)Man(9)GlcNAc(2).
