Proteome-Wide Analysis of Single-Nucleotide Variations in the N-Glycosylation Sequon of Human Genes

N-linked glycosylation is one of the most frequent post-translational modifications of proteins with a profound impact on their biological function. Besides other functions, N-linked glycosylation assists in protein folding, determines protein orientation at the cell surface, or protects proteins from proteases. The N-linked glycans attach to asparagines in the sequence context Asn-X-Ser/Thr, where X is any amino acid except proline. Any variation (e.g. non-synonymous single nucleotide polymorphism or mutation) that abolishes the N-glycosylation sequence motif will lead to the loss of a glycosylation site. On the other hand, variations causing a substitution that creates a new N-glycosylation sequence motif can result in the gain of glycosylation. Although the general importance of glycosylation is well known and acknowledged, the effect of variation on the actual glycoproteome of an organism is still mostly unknown. In this study, we focus on a comprehensive analysis of non-synonymous single nucleotide variations (nsSNV) that lead to either loss or gain of the N-glycosylation motif. We find that 1091 proteins have modified N-glycosylation sequons due to nsSNVs in the genome. Based on analysis of proteins that have a solved 3D structure at the site of variation, we find that 48% of the variations that lead to changes in glycosylation sites occur at the loop and bend regions of the proteins. Pathway and function enrichment analysis show that a significant number of proteins that gained or lost the glycosylation motif are involved in kinase activity, immune response, and blood coagulation. A structure-function analysis of a blood coagulation protein, antithrombin III and a protease, cathepsin D, showcases how a comprehensive study followed by structural analysis can help better understand the functional impact of the nsSNVs

IPA results for functional analysis of the protein data set.

<p>The threshold for all the analyses was set to p<0.05 and the data is plotted against –log of p values. The top three categories with lowest p value are presented for three IPA categories: <b>A</b>. molecular and cellular functions; CTCSI -Cell to Cell Signaling and Interaction, CM- Cellular Movement, CFM- Cellular Function and Maintenance. <b>B</b>. physiological system development and functions; TD- Tissue Development, OD- Organismal Development HSDF- Hematological System Development and Function. <b>C</b>. canonical pathways; CS- Coagulation System, IPAP- Intrinsic Prothrombin Activation Pathway, CES- Caveolar mediated Endocytosis Signaling.</p

Frequency of amino acids that are part of the glycosylation motif.

<p>Frequency of amino acids that are part of the glycosylation motif.</p

Cellular component that the protein occupies as defined in UniProtKB/Swiss-Prot keyword¹.

1<p>The same proteins can have more than one keyword if it is found to be present in more than one location.</p>2<p>15293 out of the total 20242 proteins in the human proteome have a Cellular Component keyword.</p>3<p>935 out 1091 nsSNV glycoproteins have a Cellular Component keyword.</p

Summary of results of loss and gain of N-glycosylation in the human proteome.

<p>Summary of results of loss and gain of N-glycosylation in the human proteome.</p

Network of the interactions of secreted proteins.

<p>Multiplicity of lines connecting individual proteins indicates the level of evidence for the interactions. A green line - neighborhood evidence; a blue line - concurrence evidence; a purple line - experimental evidence; a light blue line - database evidence; a black line – co-expression evidence. Antithrombin III (SERPINC1) is boxed in red and the interacting partners are marked with red arrows.</p

Effect of loss of N-linked glycosylation in cathepsin D due to nsSNV.

<p><b>A</b>. Crystal structure of cathepsin D (PDB id: 1LYA) in complex with a bound N-linked oligosaccharide. The glycosylation motif extends from a loop to a strand with the N in the loop and the remainder in the strand. The interacting residues in pink from Chain A (134NGT136) and N209 from Chain B are shown as sticks. Oxygen atoms are colored in red and nitrogens in blue. The oligosaccharide (NAG-NAG-BMA-MAN) forms a hydrogen bond with N134, T136 and N209. <b>B</b>. Crystal structure of cathepsin D (PDB id: 1LYA) depicting the variation at position 136 from T to an A. There is a change in the charge distribution resulting in a loss of a hydrogen bond. This may result in the loss of the glycosylation site. A and B within parenthesis represent the chain Ids within the PDB.</p