Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes

Abstract

Conformational aspects of N-glycosylation have been investigated with a series of proline-containing peptides as molecular probes. The results demonstrate that, depending on the position of the imino acid in the peptide chain, dramatic alterations of glycosylation rates are produced, pointing to a critical contribution of the amino acids framing the 'marker sequence' triplet Asn-Xaa-Thr(Ser) on the formation of a potential sugar-attachment site. No glycosyl transfer at all was detectable to those peptides containing a proline residue either in position Xaa or in the next position beyond the threonine of the Asn-sequon on the C-terminal side, whereas the hexapeptide Pro-Asn-Gly-Thr-Ala-Val was glycosylated at a high rate. (Emboldened residues denote the 'marker sequence' that is identical in all the peptides; italicized residues distinguish the positions of proline in the various peptides.) Studies with space-filling models reveal that the lack of glycosyl-acceptor capabilities of Ala(Pro)-Asn-Gly-Thr-Pro-Val might be directly related to their inability to adopt and/or stabilize a turn or loop conformation which permits the catalytically essential interaction between the hydroxy amino acid and the asparagine residue within the 'marker sequence' [Bause & Legler (1981) Biochem. J. 195, 639-644]. This conclusion is supported by circular-dichroism spectroscopic data, which suggest structure-forming potentials in this type of non-acceptor peptides dominating over those that favour the induction of an appropriate sugar-attachment site in the acceptor peptides. The lack of acceptor properties of Tyr-Asn-Pro-Thr-Ser-Val indicates that even small modifications in the 'recognition' pattern are not tolerated by the N-glycosyltransferases.

The mouse prostaglandin E receptor EP2 subtype: cloning, expression, and Northern blot analysis

“…Analysis of the predicted amino acid sequence indicated that it possesses seven hydrophobic segments typical of guanine nucleotide-binding protein-coupled rhodopsin-type receptors, and is 86% identical to that of the human EP2 receptor [11]. Two potential N-glycosylation sites [18] were found in the N-terminal and the first extracellular loop regions. Five potential sites of phosphorylation by protein kinase C [19] were found in the second and third intracellular loops and the C-terminal regions.…”

Structural features of the McPC603 Fab fragment not defined in the X‐ray structure

“…While there is another Asn-X-Thr site at position Asn H127, the middle amino acid X is proline, and this prevents N-glycosylation [22]. To eliminate the possibility of an additional O-linked glycosylation, which had been found in another mouse IgA at the position corresponding to Ser H223 [23], as the cause of the remaining discrepancy between the molecular weight of the recombinant and the Nglycosidase treated Fat, fragment, an amino sugar analysis was carried out as described in section 2.…”

Solid-phase extraction of N-linked glycopeptides

“…A PeptideProphet probability score of Z0.9 is used as a filter to remove low-probability peptide identifications. As it is known that the majority of N-linked glycosylation occurs at a consensus N-X-S/T sequon (where X is any amino acid except proline) 9 , the assigned peptide sequences from…”

“…In addition, the number of N-linked glycosylation sites in the human extracellular proteome is modest and is identifiable with current proteomic technology. For example, N-glycosites, which generally contain the N-X-S/T sequence motif (where X denotes any amino acid except proline) 9 , are found in just 3% of tryptic peptides from the entire human proteome; yet they represent the majority of extracellular proteins (over 70%) (see ref. 10).…”