Abstract
O-linked glycosylation is an important post-translational modification of mucin-type protein, changes to which are important biomarkers of cancer. For this study of the enzymes of O-glycosylation, we developed a shorthand notation for representing GalNAc-linked oligosaccharides, a method for their graphical interpretation, and a pattern-matching algorithm that generates networks of enzyme-catalysed reactions. Software for generating glycans from the enzyme activities is presented, and is also available online. The degree distributions of the resulting enzyme-reaction networks were found to be Poisson in nature. Simple graph-theoretic measures were used to characterise the resulting reaction networks. From a study of in-silico single-enzyme knockouts of each of 25 enzymes known to be involved in mucin O-glycan biosynthesis, six of them, β-1,4-galactosyltransferase (β4Gal-T4), four glycosyltransferases and one sulfotransferase, play the dominant role in determining O-glycan heterogeneity. In the absence of β4Gal-T4, all Lewis X, sialyl-Lewis X, Lewis Y and Sda/Cad glycoforms were eliminated, in contrast to knockouts of the N-acetylglucosaminyltransferases, which did not affect the relative abundances of O-glycans expressing these epitopes. A set of 244 experimentally determined mucin-type O-glycans obtained from the literature was used to validate the method, which was able to predict up to 98% of the most common structures obtained from human and engineered CHO cell glycoforms.
Highlights
Glycosylation is a major post-translational modification of proteins, in which a carbohydrate moiety, called a glycan, is covalently attached to an amino acid of the polypeptide, to form a glycoprotein [1]
Our objective being to model the enzymes of mucin-type O-linked glycosylation, we first developed a model language to represent O-glycan structures succinctly in linear string form, to which a set of pattern-matching rules was applied to simulate the activities of a set of 25 glycosyltransferase and sulfotransferase enzymes
We studied the effects of single-enzyme knockouts on the properties of the PLOS Computational Biology | DOI:10.1371/journal.pcbi
Summary
Glycosylation is a major post-translational modification of proteins, in which a carbohydrate moiety, called a glycan, is covalently attached to an amino acid of the polypeptide, to form a glycoprotein [1]. Glycans are formed by the sequential addition of monosaccharides from nucleotide-sugar donors to the glycoprotein acceptor, a process that is catalysed by glycosyltransferase enzymes, which are located in the endoplasmic reticulum and Golgi apparatus. Mucins are a class of large glycoproteins that contain a large number of Ser/Thr in close proximity, which can be heavily O-glycosylated. The initial step of mucin-type glycosylation is the attachment of a GalNAc (N-acetyl-D-galactosamine) to an unoccupied Ser/Thr on the protein acceptor. In the innate immune response, cell-cell recognition is dependent on the expression of a number of different carbohydrate epitopes on carrier proteins, which include both sulfated and non-sulfated versions of Lewis X (Lex), Lewis A (Lea), Lewis B (Leb) [9] and, more rarely, Lewis Y (Ley) antigens [10]
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