Abstract
Polysialylation is the enzymatic addition of a highly negatively charged sialic acid polymer to the non-reducing termini of glycans. Polysialylation plays an important role in development, and is involved in neurological diseases, neural tissue regeneration, and cancer. Polysialic acid (PSA) is also a biodegradable and non-immunogenic conjugate to therapeutic drugs to improve their pharmacokinetics. PSA chains vary in length, composition, and linkages, while the specific sites of polysialylation are important determinants of protein function. However, PSA is difficult to analyse by mass spectrometry (MS) due to its high negative charge and size. Most analytical approaches for analysis of PSA measure its degree of polymerization and monosaccharide composition, but do not address the key questions of site specificity and occupancy. Here, we developed a high-throughput LC-ESI-MS/MS glycoproteomics method to measure site-specific polysialylation of glycoproteins. This method measures site-specific PSA modification by using mild acid hydrolysis to eliminate PSA and sialic acids while leaving the glycan backbone intact, together with protease digestion followed by LC-ESI-MS/MS glycopeptide detection. PSA-modified glycopeptides are not detectable by LC-ESI-MS/MS, but become detectable after desialylation, allowing measurement of site-specific PSA occupancy. This method is an efficient analytical workflow for the study of glycoprotein polysialylation in biological and therapeutic settings.
Highlights
The enzymatic addition of carbohydrates to a protein, is a common posttranslational modification that plays a variety of physiological roles.1 Protein glycosylation is critical in protein folding and transport through the secretory pathway, and is important for protein solubility and stability, cell-cell interactions, and ligand-receptor binding.1-4 N-linked glycosylation occurs at selected asparagine (N) residues within the consensus “sequon” N-X-S/T (where X ≠ proline).5 N-linked glycans are pre-assembled on the membrane of the endoplasmic reticulum (ER) as a dolichol-linked Glc[3]Man[9]GlcNAc[2] structure (Glc, glucose; Man, mannose; and GlcNAc, N-Acetylglucosamine), and are transferred en bloc by the enzyme oligosaccharyltransferase to the polypeptide in the lumen of the ER.3 Glycans are modified as proteins traverse the secretory pathway, leading to heterogeneity in the glycan structures in mature glycoproteins.1, 6, 7 The presence and structures of glycans at particular sites have a major influence on protein conformation, stability, and function.8
Endosialidase NF (EndoNF) is a tail-spike protein from bacteriophage K1F that cleaves Polysialic acid (PSA) leaving an oligosialic acid chain attached to the glycan.37 recombinant soluble human NCAM variant (rHuNCAM) showed anti-PSA reactivity (Figure 3, lane 4) that was lost after EndoNF treatment (Figure 3, lane 5), while there was anti-neural cell adhesion molecule (NCAM) antibody reactivity in both samples (Figure 3, lanes 6 and 7)
We observed a reduction in the apparent molecular weight of rHuNCAM after EndoNF treatment by Coomassie Blue stained SDS-PAGE (Figure 3, lanes 2 and 3)
Summary
The enzymatic addition of carbohydrates to a protein, is a common posttranslational modification that plays a variety of physiological roles.1 Protein glycosylation is critical in protein folding and transport through the secretory pathway, and is important for protein solubility and stability, cell-cell interactions, and ligand-receptor binding.1-4 N-linked glycosylation occurs at selected asparagine (N) residues within the consensus “sequon” N-X-S/T (where X ≠ proline).5 N-linked glycans are pre-assembled on the membrane of the endoplasmic reticulum (ER) as a dolichol-linked Glc[3]Man[9]GlcNAc[2] structure (Glc, glucose; Man, mannose; and GlcNAc, N-Acetylglucosamine), and are transferred en bloc by the enzyme oligosaccharyltransferase to the polypeptide in the lumen of the ER.3 Glycans are modified as proteins traverse the secretory pathway, leading to heterogeneity in the glycan structures in mature glycoproteins.1, 6, 7 The presence and structures of glycans at particular sites have a major influence on protein conformation, stability, and function.8. This workflow combines mild acid hydrolysis to remove PSA from glycans, followed by LC-ESI-MS/MS for detection and site-specific relative quantification of previously polysialylated asialoglycopeptides (Figure 1).
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