N-linked glycans of the human insulin receptor and their distribution over the crystal structure.

The human insulin receptor (IR) homodimer is heavily glycosylated and contains a total of 19 predicted N-linked glycosylation sites in each monomer. The recent crystal structure of the IR ectodomain shows electron density consistent with N-linked glycosylation at the majority of sites present in the construct. Here, we describe a refined structure of the IR ectodomain that incorporates all of the N-linked glycans and reveals the extent to which the attached glycans mask the surface of the IR dimer from interaction with antibodies or other potential therapeutic binding proteins. The usefulness of Fab complexation in the crystallization of heavily glycosylated proteins is also discussed. The compositions of the glycans on IR expressed in CHO-K1 cells and the glycosylation deficient Lec8 cell line were determined by protease digestion, glycopeptide purification, amino acid sequence analysis, and mass spectrometry. Collectively the data reveal: multiple species of complex glycan at residues 25, 255, 295, 418, 606, 624, 742, 755, and 893 (IR-B numbering); multiple species of high-mannose glycan at residues 111 and 514; a single species of complex glycan at residue 671; and a single species of high-mannose glycan at residue 215. Residue 16 exhibited a mixture of complex, hybrid, and high-mannose glycan species. Of the remaining five predicted N-linked sites, those at residues 397 and 906 were confirmed by amino acid sequencing to be glycosylated, while that at residue 78 and the atypical (NKC) site at residue 282 were not glycosylated. The peptide containing the final site at residue 337 was not recovered but is seen to be glycosylated in the electron density maps of the IR ectodomain. The model of the fully glycosylated IR reveals that the sites carrying high-mannose glycans lie at positions of relatively low steric accessibility.

The location and characterisation of the O-linked glycans of the human insulin receptor.

O-linked glycosylation is a post-translational and post-folding event involving exposed S/T residues at beta-turns or in regions with extended conformation. O-linked sites are difficult to predict from sequence analyses compared to N-linked sites. Here we compare the results of chemical analyses of isolated glycopeptides with the prediction using the neural network prediction method NetOGlyc3.1, a procedure that has been reported to correctly predict 76% of O-glycosylated residues in proteins. Using the heavily glycosylated human insulin receptor as the test protein six sites of mucin-type O-glycosylation were found at residues T744, T749, S757, S758, T759, and T763 compared to the three sites (T759 and T763- correctly, T756- incorrectly) predicted by the neural network method. These six sites occur in a 20 residue segment that begins nine residues downstream from the start of the insulin receptor beta-chain. This region which also includes N-linked glycosylation sites at N742 and N755, is predicted to lack secondary structure and is followed by residues 765-770, the known linear epitope for the monoclonal antibody 18-44.

Structure of AMA1 from Plasmodium falciparum reveals a clustering of polymorphisms that surround a conserved hydrophobic pocket.

Apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate that possesses polymorphisms that may pose a problem for a vaccine based on this antigen. Knowledge of the distribution of the polymorphic sites on the surface of AMA1 is necessary to obtain a detailed understanding of their significance for vaccine development. For this reason we have sought to determine the three-dimensional structure of AMA1 using x-ray crystallography. The central two-thirds of AMA1 is relatively conserved among Plasmodium species as well as more distantly related apicomplexan parasites, and contains two clusters of disulfide-bonded cysteines termed domains I and II. The crystal structure of this fragment of AMA1 reported here reveals that domains I+II consists of two intimately associated PAN domains. PAN domain I contains many long loops that extend from the domain core and form a scaffold for numerous polymorphic residues. This extreme adaptation of a PAN domain reveals how malaria parasites have introduced significant flexibility and variation into AMA1 to evade protective human antibody responses. The polymorphisms on the AMA1 surface are exclusively located on one side of the molecule, presumably because this region of AMA1 is most accessible to antibodies reacting with the parasite surface. Moreover, the most highly polymorphic residues surround a conserved hydrophobic trough that is ringed by domain I and domain II loops. Precedents set by viral receptor proteins would suggest that this is likely to be the AMA1 receptor binding pocket.

Refolding, purification, and crystallization of apical membrane antigen 1 from Plasmodium falciparum.

Extracellular domains of malaria antigens almost invariably contain disulphide linkages but lack N- and O-linked glycosylation. The best practical approach to generating recombinant extracellular Plasmodium proteins is not established and the problems encountered when using a bacterial expression/refolding approach are discussed in detail. Limited proteolysis experiments were used to identify a relatively non-flexible core region of the Plasmodium falciparum protein apical membrane antigen 1 (AMA1), and refolding/purification was used to generate two fragments of AMA1. Several chromatographically distinct AMA1 variants were identified that are presumably differentially refolded proteins. One of these AMA1 preparations proved to be crystallizable and generated two crystal forms that diffracted X-rays to 2 A resolution.