Post-translational modification by glycosylation

Post-translational modifications (PTMs) represent one of the major sources of protein structure diversity affecting protein conformation, charge and hydrophobicity, either by covalent addition of groups to amino acid side chains or by proteolytic cleavage of one or more peptide bonds in a protein by proteases. There are more than 200 kinds of covalent modifications, among which phosphorylation, glycosylation, ubiquitination, acetylation, oxidation represent few of the most analyzed PTMs, resulting in a large number of publication entries in PubMed every year. An overview of several most common PTMs in given in Appendix 2. Covalent modifications have crucial impact on a protein’s three dimensional structure, folding, activity, cellular localization, and are involved in protein-protein interactions and signal transduction pathways. Knowledge of the attachment site of PTMs is of major importance for the understanding of protein function and its regulation in biological pathways. Characterization of modifications represents a formidable analytical challenge, primarily derived from the fact that some PTMs are present at substoechiometric level, while others are highly heterogeneous. In the past years, mass spectrometry proved to be the method of choice for microcharacterization of PTMs, due to the high sensitivity, low sample consumption and its ability to resolve complex mixtures.

Several decades ago, following the discovery of the genetic code, it became clear that carbohydrates represent integral components of a broad range of molecules, collectively termed glycoconjugates. These comprise compounds in which sugar chains (glycans) are covalently attached to either polypeptide or lipid chains to form glycoproteins, proteoglycans and glycolipids. Most cell-surface and secreted proteins are glycosylated – a fact that impacts on efforts to understand the biological relevance of specific protein expression and modification patterns. Several significant biological roles of carbohydrates include location of a protein within the cell, protection of a protein against proteolytic attack, control of the lifetime of circulating cells and glycoproteins, induction and maintenance of the spatial conformation in a biologically active form, facilitation of the extracellular secretion, as well as modulation of the immune response. Surface

carbohydrates serve as the interface between the cell and its environment, and define self versus non-self. Depending on the type of the linkage atom to which carbohydrates are covalently attached, glycans are categorized as N-, O-, C- and S-glycans. Unlike the core proteins, glycans are expressed as a set of variations on a core structure and are polydisperse in nature. Figure 2.29 shows the most common monosaccharide building blocks found in N- and O-linked glycans, with the abbreviations and symbols used throughout the following chapters.

Figure 2.29: Chair conformation of the most common monosaccharide building blocks contained in N- and O-linked glycans.

Prediction of glycoproteins is difficult and challenging, as no consensus sequence has been defined thus far, e.g. for O-glycosylation. It has been found that N-glycosylation requires the consensus sequence Asn-Xxx-Ser/Thr/Cys, where Xxx can be any amino acid, except for Pro. Protein modification by N-glycosylation is initiated in the endoplasmic reticulum (ER) by the attachment of a conserved oligosaccharide precursor containing two N-acetyl glucosamine, nine mannose and three glucose residues (Glc3Man9GlcNAc2) to the Asn side chain of the nascent polypeptide. A large orchestra of enzymes including glycosidases and glycosyl transferases trim down and elongate the precursor glycan, already in ER and with potential continuation in the Golgi apparatus, such that three major types of mature N-glycans may be synthesized, as follows: (i) high mannose, consisting primarily of Man with a maximum number of nine mannoses possible unless not fully processed, (ii) complex type glycans, mainly composed of GlcNAc and Gal with or without

sialic acid, where a fucose may be added to the first GlcNAc in the core; and (iii) hybrid type glycans which are composed of mannose, GlcNAc-Gal and with or without sialic acids (Figure 2.30). High mannose glycans are commonly found in invertebrates, e.g. in glycoproteins forming viral envelopes, whereas only vertebrates appear to be capable of synthesizing hybrid and complex N-glycans, in addition to the high mannose.

Figure 2.30: Pathways for biosynthesis of N-linked glycans: a precursor glycan with the composition Glc3Man9GlcNAc2 (middle) is attached to the asparagine side chain at the consensus sequence (Asn-Xxx-Ser/Thr) during ribosomal protein biosynthesis. Enzymatic processing of the precursor glycan leads to diversification of the N-linked species into high mannose (top left), hybrid (top right) and complex type glycans (bottom left), while a minimal pentasaccharide core of composition Man3GlcNAc2, highlighted with grey, remains common to all structures. Anomeric forms and linkages between monosaccharides are indicated in black Greek letters and numbers, respectively. Colour code: green rectangle – N-acetyl glucosamine, yellow circle – galactose, blue circle – mannose, green circle – glucose, red triangle – fucose, and purple rhombus – N-acetyl neuraminic acid.

Biosynthesis of O-glycans is more complex than that of N-glycans and does not require a precursor glycan or a consensus of amino acids. Several hundreds of different O-glycan structures have been described, yet little is known about their specific functions. The tremendous structural variation is due to the fact that at least eight different core structures of O-glycans exist in mammalian mucins. Sugars are transferred to the Ser and/or Thr side chain and subsequently elongated from specific nucleotide sugar donors by the action of specific membrane-bound glycosyl transferases in the Golgi apparatus. In cancer cells, many of the enzymes involved in O-glycan biosynthesis are up- or down-regulated. The

initial step in O-glycan biosynthesis is the attachment of N-acetyl galactosamine (GalNAc), representing the only core structure common to all O-linked oligosaccharides (Figure 2.31).

Figure 2.31: Principle pathways of biosynthesis of O-glycans, initiated by the attachment of N-acetyl galactosamine (yellow rectangle) to the side chain of Ser or Thr. Elongation and branching of this substrate by the action of various sugar transferases results in formation of Core I, Core II, Core III and Core IV glycans, which can be further elongated with (GlcNAc-Gal) disaccharide repeat units and terminated by N-acetyl neuraminic acid. Anomeric forms and linkages between monosaccharides are indicated in red Greek letters and numbers, respectively. Colour code: yellow rectangle – N-acetyl galactosamine, green rectangle – N-acetyl glucosamine, and yellow circle – galactose.

Consequently, one glycosylation site may have multiple glycan structures (microheterogeneity), while a protein may contain multiple glycosylation sites (macroheterogeneity). Glycosylation increases the complexity of protein molecules and causes them to migrate as diffuse bands or spots on SDS-PAGE gels to complicate efforts to identify protein expression patterns that correlate with disease state. Whereas unmodified proteins can often be studied by X-ray crystallography or NMR spectroscopy, these methods fail to provide satisfactory results with glycoproteins, as a result of the increased heterogeneity derived from glycosylation.