Mechanisms in Protein O-Glycan Biosynthesis andClinical and Molecular Aspects of ProteinO-Glycan Biosynthesis Defects: A ReviewSuzan Wopereis,1Dirk J. Lefeber,1E´va Morava,2and Ron A. Wevers1*Background: Genetic diseases that affect the biosynthe-sis of protein O-glycans are a rapidly growing group ofdisorders. Because this group of disorders does not havea collective name, it is difficult to get an overview ofO-glycosylation in relation to human health and dis-ease. Many patients with an unsolved defect in N-glycosylation are found to have an abnormal O-glyco-sylation as well. It is becoming increasingly evident thatthe primary defect of these disorders is not necessarilylocalized in one of the glycan-specific transferases, butcan likewise be found in the biosynthesis of nucleotidesugars, their transport to the endoplasmic reticulum(ER)/Golgi, and in Golgi trafficking. Already, disordersin O-glycan biosynthesis form a substantial group ofgenetic diseases. In view of the number of genes in-volved in O-glycosylation processes and the increasingscientific interest in congenital disorders of glycosyla-tion, it is expected that the number of identified dis-eases in this group will grow rapidly over the comingyears.Content: We first discuss the biosynthesis of proteinO-glycans from their building blocks to their secretionfrom the Golgi. Subsequently, we review 24 differentgenetic disorders in O-glycosylation and 10 different ge-netic disorders that affect both N- and O-glycosylation.The key clinical, metabolic, chemical, diagnostic, and ge-netic features are described. Additionally, we describemethods that can be used in clinical laboratory screeningfor protein O-glycosylation biosynthesis defects and theirpitfalls. Finally, we introduce existing methods that might beuseful for unraveling O-glycosylation defects in the future.© 2006 American Association for Clinical ChemistryThe human proteome, originating from expression of theprotein-coding genes of the genome, comprises ⬃30 000proteins (1), a surprisingly low number considering thatthe genome of the nematode Caenorhabditis elegans com-prises 20 000 genes (2 ). However, a higher order ofcomplexity of protein products in humans arises frompretranslational events, such as alternative splicing, andposttranslational modifications, such as phosphorylationand glycosylation. Glycosylation, the enzymatic additionof carbohydrates to proteins or lipids, is the most commonand most complex form of posttranslational modification.This is illustrated by the estimation that 1% of human1Laboratory of Pediatrics and Neurology and2Department of Pediatrics,Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.* Address correspondence to this author at: Laboratory of Pediatrics andNeurology (830), Institute of Neurology, Radboud University Nijmegen Med-ical Center, Geert Grooteplein 10, 6525 GA Nijmegen, The Netherlands. Fax31-24-3540297; e-mail [email protected] November 2, 2005; accepted January 24, 2006.Previously published online at DOI: 10.1373/clinchem.2005.0630403Nonstandard abbreviations: hLys, hydroxylysine; CDG, congenital dis-orders of glycosylation; GalNAc, N-acetylgalactosamine; NeuAc, N-acetyl-neuraminic acid (sialic acid); GlcNAc, N-acetylglucosamine; sLex, sialyl Lewisxantigen; GAG, glycosaminoglycan; GlcA, glucuronic acid (or glucuronate);EGF, epidermal growth factor; TSR, thrombospondin type-1 repeat; ER,endoplasmic reticulum; GNE/MNK, UDP-GlcNAc 2 epimerase/N-acetylman-nosamine kinase; Dol-P, dolichol phosphate; NST, nucleotide sugar trans-porter; CHO, Chinese hamster ovary; FUCT, GDP-Fuc transporter;␤3-Gal-T,␤3-galactosyltransferase; Cosmc, core 1␤3-Gal-T-specific molecular chaper-one; pp-GalNAc-T, polypeptide N-acetylgalactosaminyltransferase; EXTL ex-ostoses-like; COP, coatomer protein; ERGIC, endoplasmic reticulum-Golgiintermediate compartment; SNARE, soluble N-ethylmaleimide-sensitive fu-sion attachment protein receptor; COG, conserved oligomeric Golgi complex;GalNT, N-acetylgalactosyltransferase; FTC, familial tumoral calcinosis;B4GalT,␤-1,4-galactosyltransferase; HME, hereditary multiple exostoses;MCD, macular corneal dystrophy; SED, spondyloepiphyseal dysplasia;DTDST, diastrophic dysplasia sulfate transporter; DTD, diastrophic dysplasia;ACGB1, achondrogenesis type 1B; AO-II, atelosteogenesis type II; EDM4,multiple epiphyseal dysplasia 4; PAPSS2, 3⬘-phosphoadenosine 5⬘-phospho-sulfate synthase 2; APS, adenosine 5⬘-phosphosulfate; PAPS, 3⬘-phosphoad-enosine 5⬘-phosphosulfate; WWS, Walker–Warburg syndrome; LGMD2, limb-girdle muscular dystrophy type 2; MEB, muscle– eye–brain disease; FCMD,Fukuyama-type congenital muscular dystrophy; FKRP, fukutin-related pro-tein; MDC, congenital muscular dystrophy; LARGE, N-acetylglucosaminyl-like protein; hIBM, hereditary inclusion body myopathy; DMRV, distal myop-athy with rimmed vacuoles; FUT, fucosyltransferase; IEF, isoelectric focusing;apoC-III, apolipoprotein C-III; and CMRD, chylomicron retention disease.Clinical Chemistry 52:4574– 600 (2006)Reviews574genes are required for this specific process (3 ). Further-more, more than one half of all proteins are glycosylated,according to estimates based on the SwissProt database(4). In humans, protein-linked glycans can be divided into3 categories: N-linked (linkage to the amide group ofAsn), O-linked [linkage to the hydroxyl group of Ser, Thr,or hydroxylysine (hLys)3], and C-linked (linkage to acarboxyl group of Trp) (5).Initially, the study of glycoproteins and their role inhuman congenital diseases focused on N-linked glycans.The diseases in this pathway have collectively been re-ferred to as congenital disorders of glycosylation (CDG).N-Glycans share a common protein– glycan linkage andhave a common biosynthetic pathway that diverges onlyin the late Golgi stage. Endoglycosidases are available thatcan cleave intact N-glycans from the protein backbone,making it relatively easy to study alterations of N-glyco-sylation in health and disease. In contrast, O-glycans arebuilt on different protein glycan linkages and have ex-tremely diverse structures; in addition, there is no en-doglycosidase available for the release of intact O-glycans.However, methods for the chemical release of O-glycanshave been developed and have enabled the generation ofstructural information for O-glycans, making it morefeasible to study alterations in O-glycosylation in relationto health and disease. This review