Altered glycosyltransferases in colorectal cancer

The cellular glycome, comprised of the entire spectrum of glycan structures, is immensely complex and has gained increasing interest due to its ability to influence and regulate key cellular processes like cell adhesion, migration, cell–cell recognition, and immune surveillance [1]. Glycosylation, the addition of defined sugars to proteins and lipids, occurs in the endoplasmic reticulum or the Golgi apparatus [2] and is catalyzed by the collective action of a family of enzymes (glycosyltransferases) that transfer sugar residues from nucleotidesugar donors to lipid’ protein or sugar acceptors, and enzymes (glycosidases) that catalyze the hydrolysis of glycosidic linkages [3,4]. The process is highly substrate-, site-, and contextspecific, and leads to the formation of highly complex glycan structures that can be distinguished as N-linked or O-linked based on addition of the sugars onto side chains of Asn or Ser/ Thr residues, respectively [4,5]. A multitude of glycosyltransferase enzymes catalyze the different glycan biosynthetic pathways, resulting in the formation of a glycocode that bears key information for critical cellular functions involved in biologic homeostasis [6]. Of note, a single glycosyltransferase can often be involved in a variety of glycosylation sub-network pathways [1]. Aberrations in Nand/or O-linked glycosylation can perturb the cellular glycan structures and severely hinder the normal functioning of proteins and lipids involved in critical biological processes [4,5]. Indeed, altered Nand/or O-linked glycosylation is a hallmark of several cancers, including colorectal cancer (CRC), that promote tumor progression and metastasis [4,5]. While aberrant glycosylation patterns have led to the identification of several cancer-associated antigens many of which are glycoconjugates [4], the molecular basis for aberrant glycosylation in cancer and the underlying mechanisms by which they contribute to tumor progression, however, remain poorly understood. Some of the causative factors underlying altered protein glycosylation in cancers include aberrant expression levels of glycosyltransferases, altered localization of glycosyltransferases within subcellular compartments, bioavailability of protein substrates, and/or the absence of necessary cofactors that aid in normal glyco-biogenesis [1,4,5]. In particular, our previous studies in familial and sporadic CRCs identified functionally inactivating germline and somatic mutations in the gene encoding for GALNT12 (Polypeptide N-acetylgalactosaminyltransferase 12) [7], suggesting that aberrant glycosylation in cancers could stem from genetic defects in the glycosyltransferase genes. These findings prompted us to undertake an in-depth characterization of CRC-associated mutational alterations in genes encoding glycosylation enzymes using targeted nextgeneration sequencing [8]. Of the 430 glycosylation-associated genes assessed, 36 genes exhibited mutational alterations (missense, nonsense, splice site, and frameshift mutations) in CRCs, with 12 genes displaying significantly higher mutation rates than the expected background rate in CRCs [8]. Interestingly, further enrichment analysis of the 12 genes showed that three genes, B3GNT2 (β-1,3-N Acetylglucosaminyltransferase 2), B4GALT2 (β-1,4-Galactosyltransferase 2), and ST6GALNAC2 (αN-Acetylgalactosaminide α-2,6-Sialyltransferase 2), belonged to polylactosamine and Nand O-glycosylation pathway [8]. While B3GNT2 and B4GALT2 are involved in polyLacNAc biosynthesis on N-linked tetraantennary structures and on Core 1, 2, and 3 O-glycan core structures [9–11], ST6GALNAC2 terminates glycan chain elongation by the addition of sialic acid to the peptide GalNAc of O-glycan Core 1 or Core 3 structures [10,12]. Similar to GALNT12, mutations in these three genes predominantly mapped to the catalytic domain of respective enzymes [8]. Biochemical analyses of wild-type and mutant B3GNT2, ST6GALNAC2, and B4GALT2 demonstrated significant alterations in respective glycosyltransferase function [8]. For instance, mutations in B3GNT2 led to either loss of enzymatic function or mislocalization of the protein. Genomic loss of the wild-type allele in B4GALT2 was accompanied by a missense mutation that disrupted the post-translation modification pattern of the B4GALT2 protein [8], and finally, mutant ST6GALNAC2 consistently showed hyper enzyme activity [8] as compared to its wild-type counterpart. GALNT12, B3GNT2, B4GALT2, and ST6GALNAC2 are enzymes involved in mucin type O-glycosylation and catalyze the formation of complex O-glycan structures [10]. While the endogenous substrates of these enzymes remain to be determined, given that an individual glycosyltransferase enzyme may be involved in the post-translational modification and maturation of multiple protein substrates, it is possible that genetic alterations in glycosylation pathway genes may accordingly affect a multitude of proteins, through loss-