Clinical Chemistry 52 4 574 600 2006 Reviews Mechanisms in Protein O Glycan Biosynthesis and Clinical and Molecular Aspects of Protein O Glycan Biosynthesis Defects A Review Suzan Wopereis 1 Dirk J Lefeber 1 E va Morava 2 and Ron A Wevers1 Background Genetic diseases that affect the biosynthesis of protein O glycans are a rapidly growing group of disorders Because this group of disorders does not have a collective name it is difficult to get an overview of O glycosylation in relation to human health and disease Many patients with an unsolved defect in Nglycosylation are found to have an abnormal O glycosylation as well It is becoming increasingly evident that the primary defect of these disorders is not necessarily localized in one of the glycan specific transferases but can likewise be found in the biosynthesis of nucleotide sugars their transport to the endoplasmic reticulum ER Golgi and in Golgi trafficking Already disorders in O glycan biosynthesis form a substantial group of genetic diseases In view of the number of genes involved in O glycosylation processes and the increasing scientific interest in congenital disorders of glycosylation it is expected that the number of identified diseases in this group will grow rapidly over the coming years Content We first discuss the biosynthesis of protein O glycans from their building blocks to their secretion from the Golgi Subsequently we review 24 different genetic disorders in O glycosylation and 10 different genetic disorders that affect both N and O glycosylation The key clinical metabolic chemical diagnostic and genetic features are described Additionally we describe methods that can be used in clinical laboratory screening for protein O glycosylation biosynthesis defects and their pitfalls Finally we introduce existing methods that might be useful for unraveling O glycosylation defects in the future 2006 American Association for Clinical Chemistry The human proteome originating from expression of the protein coding genes of the genome comprises 30 000 proteins 1 a surprisingly low number considering that the genome of the nematode Caenorhabditis elegans comprises 20 000 genes 2 However a higher order of complexity of protein products in humans arises from pretranslational events such as alternative splicing and posttranslational modifications such as phosphorylation and glycosylation Glycosylation the enzymatic addition of carbohydrates to proteins or lipids is the most common and most complex form of posttranslational modification This is illustrated by the estimation that 1 of human 3 Nonstandard abbreviations hLys hydroxylysine CDG congenital disorders of glycosylation GalNAc N acetylgalactosamine NeuAc N acetylneuraminic acid sialic acid GlcNAc N acetylglucosamine sLex sialyl Lewisx antigen 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 acetylmannosamine kinase Dol P dolichol phosphate NST nucleotide sugar transporter CHO Chinese hamster ovary FUCT GDP Fuc transporter 3 Gal T 3 galactosyltransferase Cosmc core 1 3 Gal T specific molecular chaperone pp GalNAc T polypeptide N acetylgalactosaminyltransferase EXTL exostoses like COP coatomer protein ERGIC endoplasmic reticulum Golgi intermediate compartment SNARE soluble N ethylmaleimide sensitive fusion 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 phosphosulfate synthase 2 APS adenosine 5 phosphosulfate PAPS 3 phosphoadenosine 5 phosphosulfate WWS Walker Warburg syndrome LGMD2 limbgirdle muscular dystrophy type 2 MEB muscle eye brain disease FCMD Fukuyama type congenital muscular dystrophy FKRP fukutin related protein MDC congenital muscular dystrophy LARGE N acetylglucosaminyllike protein hIBM hereditary inclusion body myopathy DMRV distal myopathy with rimmed vacuoles FUT fucosyltransferase IEF isoelectric focusing apoC III apolipoprotein C III and CMRD chylomicron retention disease 1 Laboratory of Pediatrics and Neurology and 2 Department of Pediatrics Radboud University Nijmegen Medical Center Nijmegen The Netherlands Address correspondence to this author at Laboratory of Pediatrics and Neurology 830 Institute of Neurology Radboud University Nijmegen Medical Center Geert Grooteplein 10 6525 GA Nijmegen The Netherlands Fax 31 24 3540297 e mail r wevers cukz umcn nl Received November 2 2005 accepted January 24 2006 Previously published online at DOI 10 1373 clinchem 2005 063040 574 575 Clinical Chemistry 52 No 4 2006 genes are required for this specific process 3 Furthermore 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 into 3 categories N linked linkage to the amide group of Asn O linked linkage to the hydroxyl group of Ser Thr or hydroxylysine hLys 3 and C linked linkage to a carboxyl group of Trp 5 Initially the study of glycoproteins and their role in human congenital diseases focused on N linked glycans The diseases in this pathway have collectively been referred to as congenital disorders of glycosylation CDG N Glycans share a common protein glycan linkage and have a common biosynthetic pathway that diverges only in the late Golgi stage Endoglycosidases are available that can cleave intact N glycans from the protein backbone making it relatively easy to study alterations of N glycosylation in health and disease In contrast O glycans are built on different protein glycan linkages and have extremely diverse structures in addition there is no endoglycosidase available for the release of intact O glycans However methods for the chemical release of O glycans have been developed and have enabled the generation of structural information for O glycans making it more feasible to study alterations in O glycosylation in relation to health and disease This review focuses on the biosynthesis of O glycans and the human congenital disorders of O glycosylation and their screening Structures of O Linked Glycans The O glycosylation