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Structural and functional characterization of the human malectin glycan-binding pocket
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The N-glycosylation pathway has several chaperones, enzymes and complexes dedicated to protein quality control and correct folding of proteins. Malectin is a membrane-anchored endoplasmic reticulum (ER) protein, which recognizes a Glc-2-N-glycan intermediate of the early steps of this pathway in nascent proteins. The high conservation of malectin in animals reveals the importance of this lectin in the ER, and recent studies point to its involvement in the ER protein quality control. The X-ray crystal structure of malectin-ligand complex revealed the molecular interaction with the D1 arm of the di-glucosylated high mannose N-glycan (Glc2Man3).
In this work, we aimed to characterize the interaction of malectin binding-pocket with its natural ligand. Therefore, to understand the molecular determinants for this highly specific interaction, we designed five specific mutants (E102A, Y104F, Y131F, D201A and Y199AY200A). Also, looking for a new binding specificity to O-glucosylation epidermal growth factor like repeats, we designed a mutation of a residue that sits in the bottom of the binding-pocket (S80Q). Structural stability studies of the malectin mutants were carried out using Differential Scanning Calorimetry analysis and their effects in the interaction with glucosylated ligands were accessed by a first screening in a polysaccharide microarray. We also analysed the binding of the wild-type malectin and the specifically designed mutants to the Glc2Man3 using the NGL-based oligosaccharide microarray. Comparison of the six proteins revealed the importance of the residues that establish interactions between the protein and the glycan. The objective of developing a protein with a new specificity was not achieved during this framework. However, the process led to the confirmation that the malectin binding pocket has adapted to perfectly accommodate the two glucose rings from the di-glucosylated N-glycan.
An important outcome from this study was to clarify the importance of several residues in the malectin specificity and, therefore, to provide more information that will contribute to the clarification of the function of this protein involved in the N-glycosylation pathway.
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Palavras-chave
Malectin Endoplasmic reticulum N-glycosylation Glycobiology carbohydrate microarrays