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Reviews |
1 Laboratory of Pediatrics and Neurology and2 Department of Pediatrics, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands.
aAddress 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{at}cukz.umcn.nl.
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Abstract |
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 Top Abstract IntroductionStructures of O-Linked GlycansBiosynthesis of O-GlycansFunctions of O-Linked GlycansCongenital Disorders in the...Screening Methods for Unraveling...References |
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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.
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Introduction |
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TopAbstractIntroductionStructures of O-Linked GlycansBiosynthesis of O-GlycansFunctions of O-Linked GlycansCongenital Disorders in the...Screening Methods for Unraveling...References |
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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 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)
1
], 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.
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Structures of O-Linked Glycans |
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TopAbstractIntroductionStructures of O-Linked GlycansBiosynthesis of O-GlycansFunctions of O-Linked GlycansCongenital Disorders in the...Screening Methods for Unraveling...References |
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or ß configuration, allowing glycan structures to form branches. Glycans therefore have a larger structural diversity in contrast to other cellular macromolecules such as proteins, DNA, and RNA, which form only linear chains. Theoretically, the 9 common monosaccharides found in humans could be assembled into more than 15 million possible tetrasaccharides, all of which would be considered relatively simple glycans (6).
The 7 different types of O-linked glycans found in humans are summarized in Table 1
. O-Linked glycans are classified on the basis of the first sugar attached to a Ser, Thr, or hLys residue of a protein. The mucin-type O-glycan, with N-acetylgalactosamine (GalNAc) at the reducing end, is the most common form in humans. In total, 8 mucin-type core structures can be distinguished, depending on the second sugar and its sugar linkage, of which cores 1–6 and core 8 have been described in humans (summarized in Table 2
) (7). In addition to the 7 core structures, the Tn (GalNAc1-Ser/Thr) and sialyl Tn [NeuAc2–6GalNAc1-Ser/Thr; where NeuAc is N-acetylneuraminic acid (sialic acid)] epitopes can be distinguished. The core structures can be further modified; for example, by the addition of an N-acetyllactosamine unit (Galß1–4GlcNAc; where GlcNAc is N-acetylglucosamine), also seen on N-glycans. The N-acetyllactosamine unit may be branched by a GlcNAcß1–6 residue or form repeating N-acetyllactosamine units, called poly N-acetyllactosamine extensions. It can also attach to the blood group determinants (A, B, and H) and the type 2 Lewis determinants [Lex, sialyl Lewisx (sLex), and Ley]. N-Acetyllactosamine elongations are seen mainly on core 2 O-glycans. Sugars occurring at the nonreducing termini include NeuAc, Fuc, GlcNAc, and GalNAc. GlcNAc and Gal residues can be modified at position 6 or at positions 3 and/or 6, respectively, by sulfation (8), and NeuAc residues can be further modified at positions 4, 7, 8, and 9 with O-acetyl ester groups (9). This gives rise to several hundreds of different mucin-type O-glycan structures, of which core 1 and 2 are most abundant (7).
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Another common type of O-glycosylation with large structural diversity involves the glycosaminoglycans (GAGs). Proteoglycans are proteins containing GAG chains. GAGs are attached to a Ser residue of a protein via the linker tetrasaccharide GlcAß1–3Galß1–3Galß1–4Xyl, except for keratan sulfate, which is linked to proteins either through N- or core 1 O-glycans. GAGs are long, unbranched polysaccharides containing a disaccharide repeat that consists of either a GalNAc or GlcNAc residue combined with a glucuronic acid (GlcA) or a Gal residue. Three different types of GAGs can be distinguished on the basis of the composition of the disaccharide repeat: (a) dermatan sulfate and chondroitin sulfate (GlcA + GalNAc); (b) heparin/heparan sulfate (GlcA + GlcNAc); and (c) keratan sulfate (Gal + GlcNAc). GlcA in dermatan sulfate and heparin/heparan sulfate can be epimerized to iduronate. The heterogeneity of GAGs results from variable O-sulfation at defined locations (10). An extra modification step occurs in heparin and heparan sulfate by the deacetylation and N-sulfation of GlcNAc residues. Regions in which the hexosamine units are acetylated remain (almost) unmodified and consist of disaccharide repeats with GlcA, whereas regions with deacetylated hexosamine units become highly sulfated and exist as disaccharide repeats with iduronate. Heparin is a highly and uniformly sulfated GAG, whereas heparan sulfate is highly sulfated only in defined blocks (11).
The structures of the other 5 O-glycan types seem to show less variability, and they occur mostly in one conformation. A frequently occurring O-linked glycan is the single GlcNAc linked to nuclear and cytosolic proteins. This posttranslational modification is more analogous to phosphorylation than to classical complex O-glycosylation because it is a reversible process catalyzed by the enzymes O-GlcNAc transferase and O-GlcNAcase, respectively (12), and the "normal glycosylation machinery" is not implicated (12)(13).
O-Galactosyl glycans have been found only on collagen domains. Gal or Glc1–2Gal residues are covalently linked to hLys residues found in collagens, but not all hLys residues become glycosylated. The collagen 3-dimensional structure depends on the extent of this posttranslational modification. The quantities and types of O-galactosyl glycans vary considerably not only among the different types of collagen, but also among the same collagen type from different tissues and even the same collagen type from different areas of the same type of tissue (14)(15).
O-Mannosyl glycans are a less common type of protein modification, present on a limited number of glycoproteins in the brain, nerves, and skeletal muscle. The best known O-mannosylglycosylated protein is -dystroglycan, which is a skeletal muscle extracellular matrix protein (16). To date, only the NeuAc2–3Galß1–4GlcNAcß1–2Man structure has been found in humans. -Dystroglycan containing Galß1–4(Fuc1–3)GlcNAcß1–2Man has been found in sheep brain (17)(18), and the O-mannosyl glycan HSO3-3GlcAß1–3Galß1–4GlcNAcß1–2Man has been detected in rat brain (18)(19). Studies have also shown that mammalian N-acetylglucosaminyltransferase IX acts on the GlcNAcß1,2-Man1-Ser/Thr moiety, suggesting that 2,6-branched O-mannosyl glycan structures are formed in the brain (20). It is therefore likely that structural diversity of O-mannosyl glycans will also be present in humans.
O-Glucosyl and O-fucosyl glycans are also rare types of protein glycosylations that have been found in the epidermal growth factor homology regions (EGF modules) of some human proteins. An EGF module is a common structural motif found in several secreted and cell-surface proteins that is often involved in mediating protein–protein interactions. The EGF repeat is typically 30–40 amino acids long and is characterized by 6 conserved Cys residues participating in 3 disulfide bridges. Glc is linked to the Ser residue in proteins in the putative consensus sequence C1XSXPC2 (where C1 and C2 are the first and second conserved cysteines of the EGF module, S is the modified Ser residue, and X can be any amino acid) (21). O-Linked Glc can be further elongated with 1 or 2 1–3 linked xyloses and is found on proteins such as human factor VII, factor IX, and protein Z (22)(23). All O-fucosylated glycoproteins are modified with a single O-linked Fuc residue (e.g., urinary-type plasminogen activator, tissue-type plasminogen activator, and coagulation factors VII and XII) except for coagulation factor IX, which contains O-linked Fuc that is elongated to the tetrasaccharide NeuAc2–6Galß1–4GlcNAcß1–3Fuc1-Ser/Thr. Most O-Fuc modifications on EGF repeats are found on the consensus site C2X3–5S/TC3 (where C2 and C3 are the second and third conserved cysteines of the EGF repeat, S/T is the modified Ser/Thr residue, and X can be any residue) (22). A second type of O-fucosylation has been identified. On thrombospondin type 1 repeats (TSRs), a disaccharide form of O-fucosyl glycans (Glcß1–3Fuc1-Ser/Thr) is found on the human extracellular matrix protein "thrombospondin-1" (24). TSRs are found in many extracellular proteins. A single TSR is 60 amino acids long and is characterized by conserved Cys, Trp, Ser, and Arg residues. The putative consensus sequence site for this modification is WX5CX2/3S/TCX2G (22).
o-glycan consensus sites
For most O-glycosylation types, a recognition consensus sequence for the attachment of the first sugar residue remains unknown. The exceptions are the O-Glc and O-Fuc modifications, for which putative consensus sites have been described [see above and Refs. (21),(22)]. The lack of a consensus sequence can arise from the coexistence of multiple transferases with overlapping but different substrate specificities, as seen, e.g., in mucin-type O-glycosylation, or is the result of a nonlimited consensus sequence, as seen, e.g., in O-GlcNAc modifications. Statistical studies yielded some general rules for mucin-type O-glycans and O-GlcNAc modifications, leading to the development of algorithms for the prediction of these 2 O-glycan types. These O-glycosylation prediction sites are available on the Internet. The NetOglyc 3.1 prediction server correctly predicts 76% of the glycosylated residues and 93% of the nonglycosylated residues in any protein (25).
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Biosynthesis of O-Glycans |
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TopAbstractIntroductionStructures of O-Linked GlycansBiosynthesis of O-GlycansFunctions of O-Linked GlycansCongenital Disorders in the...Screening Methods for Unraveling...References |
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biosynthesis of nucleotide sugars
Monosaccharides used for the biosynthesis of nucleotide sugars derive from dietary sources and salvage pathways. Glucose (Glc) and fructose (Fru) are the major carbon sources in humans from which all other monosaccharides can be synthesized (Fig. 1
). Series of phosphorylation, epimerization, and acetylation reactions convert them into various high-energy nucleotide sugar donors (see Fig. 1
). Nucleotide sugar biosynthesis takes place in the cytosol, except for CMP-NeuAc, which is synthesized in the nucleus (32).
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As observed in patients with CDG-Ia and CDG-Ib, aberrant glycosylation results from insufficient availability of GDP-Man. The availability of nucleotide sugars is tightly regulated. UDP-GlcNAc, for example, inhibits glutamine-fructose-6P-transaminase, which catalyzes the first step in the biosynthetic pathway of UDP-GlcNAc (33), and CMP-NeuAc inhibits UDP-GlcNAc-2-epimerase/N-acetylmannosamine kinase (GNE/MNK), which catalyzes the first 2 biosynthetic steps of CMP-NeuAc (34). Although much is known about the nucleotide sugar biosynthesis pathways and their feedback regulators, the actual cytosol and Golgi steady-state concentrations of most nucleotide sugars are unknown at present. Furthermore, because of the interconnected pathways of nucleotide sugar metabolism, the results of an individual enzyme deficiency are difficult to predict.
Several steps in the biosynthesis of nucleotide sugars require ATP; therefore, the metabolic state of the cell influences the availability of the nucleotide sugars. The tight regulation of the biosynthesis of nucleotide sugars means that alterations in a single nucleotide sugar can significantly impair glycosylation.
transport processes to generate monosaccharide donors in the er/golgi
The nucleotide sugars are biosynthesized in the cytosol, and their monosaccharides must be translocated into the lumen of the ER and/or Golgi before they can be used for the glycosylation process. Because nucleotide sugars cannot cross the membrane lipid bilayer, specific transport mechanisms are responsible for their translocation. Two transport mechanisms for the generation of monosaccharide donors in the ER/Golgi can be distinguished (Fig. 2
). The first mechanism is the entrance of Man and Glc through binding to the lipid carrier dolichol phosphate (Dol-P). To date, this transport system has been described only in the ER. Cytosolic Dol-P-Man and Dol-P-Glc synthases link GDP-Man and UDP-Glc to the cytosolic site of Dol-P by cleaving off the nucleotide moiety. A hypothetical "flippase" then mediates the turnover of the Dol-P monosaccharide from the cytoplasmic leaflet to the lumenal leaflet of the ER. Subsequently, the monosaccharides can be used by ER-located glycosyltransferases (Fig. 2A
) (35). As observed in patients with CDG-Ie who are deficient for Dol-P-Man synthase and in patients with CDG-If who have mutations in the MPDU1 gene, known to be required for efficient use of Dol-P-Man and Dol-P-Glc as donor substrates, abnormal glycosylation results from diminished Dol-P-monosaccharide transport (36)(37). In CDG-If patients, it was observed that the mannosylation of N-glycans, glycosylphosphatidylinotisol anchors, and C-mannosyl glycans was defective. Although O-mannosylation was not studied, it is likely that this is also aberrant in these patients.
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The second mechanism is the transport of nucleotide sugars through specific nucleotide sugar transporters (NSTs). NSTs belong to solute carrier family 35 and reside in the Golgi and/or ER membranes with their C- and N-terminal regions exposed to the cytosol. These NSTs are antiporters in which nucleotide sugar entry into the ER/Golgi is coupled to equimolar exit of the corresponding nucleoside monophosphate from the ER/Golgi lumen (38). The nucleotide moiety of the nucleotide sugar is the recognition feature required for initial binding to the NST, whereas the attached monosaccharide finally determines whether the entire nucleotide sugar is translocated. After entrance of the nucleotide sugar into the ER/Golgi lumen, a glycosyltransferase will transfer the monosaccharide to a glycan by cleaving off the nucleotide part. The nucleoside diphosphates are converted to dianionic nucleoside monophosphates (used for the antiporter) and inorganic phosphate by a nucleoside diphosphatase. It is postulated that inorganic phosphate exits the ER/Golgi lumen via a specific transporter (Fig. 2B
) (38). Nucleoside di- and monophosphates can inhibit the nucleotide sugar transport process and the activity of glycosyltransferases.
Some NSTs transport more than one substrate: for example, the UDP-Gal/UDP-GalNAc transporter (hereafter referred to as UDP-Gal transporter) (39), the UDP-GlcA/UDP-GalNAc/UDP-GlcNAc transporter (hereafter referred to as UDP-GlcA transporter) (40)(41), and the recently described UDP-Xyl/UDP-GlcNAc transporter (hereafter referred to as UDP-Xyl transporter) (42). In contrast, the CMP-NeuAc (43), GDP-Fuc(44), and UDP-GlcNAc transporters (45) are monospecific.
In general, the transport of a nucleotide sugar occurs in the organelle in which the corresponding glycosyltransferase is localized. Some nucleotide sugars enter only the lumen of Golgi vesicles, others enter the lumen of ER-derived vesicles, and a few enter both. It has been shown that the CMP-NeuAc, GDP-Fuc, UDP-GlcNAc, and UDP-Xyl transporters have a strict Golgi membrane localization (38)(42)(45), whereas the UDP-GlcA transporter is localized in the ER membrane (40). Experiments investigating the intraorganelle availability of nucleotide sugars have shown that UDP-Xyl and UDP-Glc can also be found in the ER, whereas UDP-GlcA and UDP-Glc can be found in the Golgi (38), suggesting that the corresponding NSTs are yet to be identified.
Galactosylceramide is galactosylated by a galactosyltransferase (UDP-galactose:ceramide galactosyltransferase) found exclusively in the ER, whereas the UDP-Gal transporter has mainly a Golgi localization. This galactosyltransferase is produced only in specialized cells, such as myelinating cells, spermatogonia, and in various epithelial cell types. The question of how an ER-resident glycosyltransferase could function without a source of substrate was answered by showing that the galactosyltransferase forms a complex with the UDP-Gal transporter (46). This led to the presence of a fraction of the UDP-Gal transporter in the ER. It is not clear whether this is attributable to the retention of the UDP-Gal transporter by the galactosyltransferase or to recycling of the UDP-Gal transporter through the cis-Golgi. In this way, a biosynthetic pathway can be established only when required (46). Recently, a second active mechanism has been found for the ER localization of the UDP-Gal transporter. The UDP-Gal transporter is produced in 2 splice forms, UGT1 and UGT2. UGT1 has a strict Golgi localization, whereas UGT2 shows dual localization in both the ER and Golgi caused by a dilysine motif (KVKGS) in its COOH terminus (47).
As observed in the case of patients with CDG-IIc who have a deficient GDP-Fuc transporter (FUCT) (48) and in a patient with CDG-IIf who has a deficient CMP-NeuAc transporter (49), abnormal glycosylation results from diminished NST function. In addition, in Chinese hamster ovary (CHO) lec8 and lec2 cells defective in UDP-Gal and CMP-NeuAc transport, respectively, 70%–90% of the glycans lacked that particular monosaccharide (38). It was also shown that the nucleotide sugar transport process depends on the continuous production of nucleoside monophosphates. Abeijon et al. (50) showed that in vitro transport of GDP-Man into the Golgi is severely decreased in a Saccharomyces cerevisiae guanosine diphosphatase-null mutant. All glycoproteins and glycolipids showed impaired mannosylation (50). This results indicates that NSTs are critical components of glycosylation pathways.
transfer of nucleotide sugars to the glycan
O-Glycans are assembled by the sequential action of several specific, membrane-bound glycosyl-, O-acetyl-, and sulfotransferases in a highly controlled fashion (8). The pathways of O-glycosylation are determined by the distinct substrate specificities of glycosyltransferases, sulfotransferases, and O-acetyltransferases. Transferases involved in O-glycan biosynthesis are localized mainly in the Golgi. Although many of these enzymes catalyze similar reactions, there is a surprisingly limited sequence homology among different classes. The Golgi glycosyltransferases described to date are all type II transmembrane proteins, with a short N-terminal cytoplasmic domain, a single hydrophobic membrane-spanning domain, and a large C-terminal catalytic domain localized in the lumen of the Golgi.
The activity of glycosyltransferases can be influenced by different factors. It is known, for example, that some of the glycosyltransferases require divalent cations, such as Mn2+ and/or Mg2+, for optimal action. In contrast to the reactions involving UDP- and GDP-nucleotide sugars, the biosynthetic steps involving CMP-NeuAc do not require these cations (51). Petrova et al. (52) showed that divalent cations react strongly with nucleotide sugars in solution, thus altering their conformation.
Furthermore, it was recently discovered that human core 1 ß3-galactosyltransferase (core 1 ß3-Gal-T), which is involved in the formation of core 1 (and core 2) mucin-type O-glycans, requires a molecular chaperone for its functioning. This molecular chaperone is called core 1 ß3-Gal-T-specific molecular chaperone (Cosmc) and is an ER-localized type II transmembrane protein that appears to be required for the proper folding of the core 1 ß3-Gal-T enzyme. In the absence of functional Cosmc, core 1 ß3-Gal-T is degraded in the proteosome (53). This raises the question of whether additional chaperones specific for other glycosyltransferases exist.
A third factor that might influence glycosyltransferase activity is the structure of the protein substrate. It is thought that the protein structure contains information for the action of specific transferases. This is seen, for example, in proteoglycans, in which the core protein dictates whether it will receive a heparan sulfate or a chondroitin sulfate chain (54), or in lysosomal enzymes, in which GlcNAc-phosphotransferase recognizes subtle motifs in the secondary structure and selectively phosphorylates the N-glycans on proteins that should reach the lysosome (55)(56). However, how proteins are recognized by glycosyltransferases remains largely unknown. Finally, glycosyltransferase activity can be dependent on heterocomplex formation. O-Mannosyltransferase activity, for example, is generated only when the genes POMT1 and POMT2 (both encoding mannosyltransferases) are coexpressed (57).
Golgi transferases can recognize a single sugar residue, a sugar sequence, or a peptide moiety, leading to variable specificity. With very few exceptions, each type of transferase is regio- and stereospecific. Glycosyltransferases involved in the linkage of monosaccharides to the protein backbone and those involved in the core processing of mucin-type O-glycans are specific and not involved in other classes of glycoconjugates, whereas most glycosyltransferases involved in the elongation, branching, and termination of glycans are not specific for one glycoconjugate class. For example, the ubiquitous 2,6-sialyltransferase ST6Gal I recognizes the N-acetyllactosamine unit and catalyzes the formation of an 2,6 linkage to terminal N-acetyllactosamine structures found on N-glycans, O-glycans, and glycosphingolipids, whereas the ß1,4-galactosyltransferase (Gal-T1) galactosylates any terminal GlcNAc residue.
The attachment of UDP-GalNAc in an linkage to the hydroxyl residue of Ser or Thr in mucin-type O-glycans is a complex and as yet not fully understood process. This transfer is catalyzed by specific UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (EC 2.4.1.41). The mammalian family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts) comprises 15 members, the 15th being discovered only recently (58)(59). It is estimated that at least 24 unique human pp-GalNAc-Ts exist on the basis of sequence homology (59). The different pp-GalNAc-Ts have overlapping but different specificities and are tissue specific (8)(59). It seems that mucin-type O-glycosylation proceeds in a hierarchical manner, because some of the characterized pp-GalNAc-Ts glycosylate only peptides that are already partly glycosylated (59). Currently, no consensus sequence has been formulated because every pp-GalNAc-T has its own specific attachment site. Only Ser and Thr residues that are exposed on the protein surface will be glycosylated, as O-glycosylation is a postfolding event. Therefore, O-glycosylation takes place mainly in coil, turn, and linker regions. Furthermore, all attachment sites have high Ser, Thr, and Pro content (25).
The biosynthesis of GAG structures differs from the "small O-linked glycans" in 2 significant ways: (a) the transferases required are all specific and not involved in other glycoconjugate classes, with the exception of chondroitin 6-sulfotransferase, keratan sulfate Gal-6-sulfotransferase, and the GlcNAc 6-O-sulfotransferase that also sulfates N-acetyllactosamine extensions (60); and (b) the mechanism of GAG chain elongation is different. Chondroitin/dermatan sulfate and heparin/heparan sulfate are synthesized on the common tetrasaccharide linker (GlcAß1–3Galß1–3Galß1–4Xyl). Chondroitin/dermatan sulfate is synthesized when GalNAc is transferred to the linkage region, whereas heparin/heparan sulfate is synthesized if GlcNAc is added first. It has been demonstrated that the human exostoses-like family (EXTL1, -2, and -3) is responsible for the heparin/heparan sulfate chain initiation with the attachment of the first and second GlcNAc residues and that the exostoses enzymes extosin-1 and -2 are the copolymerases that elongate the GAG chain with (GlcAß1–4GlcNAc1–4)n(10). Recently, chondroitin GalNAc transferases I and II and chondroitin synthetase were discovered (61)(62)(63). Chondroitin GalNAc transferases I and II are responsible for the initiation of the chondroitin/dermatan sulfate GAG chain with the attachment of the first few GalNAc residues to the linker region, whereas chondroitin synthetase acts as a copolymerase and is responsible for the elongation of chondroitin/dermatan sulfate with (GalNAcß1–4GlcAß1–3)n.
golgi traffic
The Golgi apparatus consists of several cisternae, starting from the nucleus with the cis-Golgi network, through the cis-, medial-, and trans-Golgi compartments, and ending with the trans-Golgi network, which are organized in the form of a stack. The Golgi position and organization within a cell are sustained largely through the combined efforts of a complex cytoskeletal matrix composed of microtubules, an actin–spectrin network, and intermediate filaments. The interaction between these filament systems and Golgi membranes is mediated by mechanochemical enzymes such as dyneins, kinesins, myosins, and dynamin and different structural proteins (64).
A schematic overview of the transport route from ER through the different Golgi compartments is shown in Fig. 3
[for reviews, see Refs. (65),(66)]. The journey of proteins between these compartments starts with the exit from 100–200 export sites on the ER in COPII-coated vesicles. Coatamer proteins (COPs) recognize transport signals present in the cytoplasmic tail of cargo membrane proteins for their incorporation in COPII vesicles. Three classes of ER export signals have been described to date. Most type I membrane proteins have a diacidic or dihydrophobic motif, and the type II glycosyltransferases have a [RK(X)RK] motif proximal to the transmembrane domain (67). Signals that direct soluble cargo into ER-derived vesicles are less well defined. It is thought that soluble proteins are exported from the ER in 2 ways: (a) through a passive bulk flow process; and (b) through an active receptor-mediated process relying on receptor-like proteins that attach proteins to the inner membrane of the coated vesicle (68).
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Different COPII-coated vesicles fuse to form the ER-Golgi intermediate compartment (ERGIC). From here, escaped ER proteins or misfolded proteins are transported back to the ER via COPI-coated vesicles. The ERGIC elements are transported to and fused with the cis-Golgi network. From here, both anterograde and retrograde transport is mediated via COPI-coated vesicles. Three major protein families regulate vesicle transport. The ARF and Sar1 family GTPases are involved in COPI and COPII vesicle formation, which starts with the activation of ARF and Sar1 by a nucleotide exchange factor into ARF-GTP and Sar1-GTP. ARF-GTP and Sar1-GTP recruit many additional components for the synthesis of the vesicle coat. Subsequently, the Rab family GTPases mediate vesicle targeting. The mammalian Rab protein family includes at least 63 isoforms. All cytosolic Rab proteins form a complex with the Rab guanine nucleotide dissociation inhibitor chaperone, which transports the Rab proteins to the membrane of specific Golgi compartments, where they become activated to the GTP state. Activated Rabs mediate vesicle motility and the tethering of transport intermediates to their target membranes. The third family consists of SNARE proteins, which direct vesicle fusion. Each type of transport vesicle carries a specific vesicle-SNARE (v-SNARE), which binds to a tethering-SNARE (t-SNARE) on the target membrane, producing the trans-SNARE complex. After fusion, the cargo is transferred to that specific compartment (65)(66).
The Golgi is a very dynamic organelle; it has the capacity to transform in response to specific stimuli or cellular changes. For example, the Golgi or any Golgi-like structures fragment into numerous tubular and vesicular structures when cells undergo mitosis: the ER export sites disappear, the Golgi integral membrane proteins are trapped in the ER, and Golgi peripheral proteins are retargeted to the ER or cytoplasm. After mitosis is complete, the Golgi is readily re-formed by outgrowth from the ER and fusion of the tubular and vesicular structures (69).
Until recently, the Golgi was seen as a static organelle. In this model, Golgi enzymes are retained within one Golgi cisterna, and cargo (the proteins that are transported to and processed in the Golgi) are transported through the different Golgi compartments in the anterograde direction via COPI vesicles. However, at present, the cisternal maturation model is favored. It is now believed that cargo remains in one cisterna and that this Golgi compartment traffics in the anterograde direction, whereas the Golgi enzymes traffic backward by COPI vesicles. This model is based on the experimental observations that cargo indeed remains in a specific cisterna and that COPI vesicles are enriched with Golgi enzymes (70)(71).
Given the sequential and competing nature of glycosyltransferases, the precise localization of these enzymes within the Golgi is of great importance. It is thought that glycosyltransferases are arranged in an assembly line in the Golgi, whereas early-acting transferases are localized in the cis-Golgi, intermediate-acting transferases in the medial-Golgi, and terminating transferases in the trans-Golgi. A signal targeting glycosyltransferases to a specific Golgi localization has not yet been described. Studies have indicated that glycosyltransferases from a certain Golgi compartment form high–molecular-mass complexes (72). The presence of multienzyme complexes is likely to be functionally relevant in the regulation of glycosylation and contribute to the maintenance of the steady-state localization of the Golgi glycosyltransferases (72). When Nilsson and Warren (73) re-directed a Golgi resident glycosyltransferase to the ER, another Golgi enzyme also was retained in the ER. Not all glycosyltransferases form complexes; in particular, those found in the trans-Golgi network seem to be unbound. Another factor that is likely to play a role in the targeting of glycosyltransferases is the thickness of the lipid bilayer, which increases en route to the plasma membrane. The fact that Golgi proteins have shorter transmembrane domains than do plasma membrane proteins suggests that cisternae of a specific compartment can accommodate glycosyltransferases with a transmembrane domain of matching length. However, it has been shown that some soluble forms of glycosyltransferases, which have lost their transmembrane domain, are retained in the Golgi probably as a result of being associated in complexes (70)(71). It is likely that more independent signals act together to mediate efficient Golgi localization.
conserved oligomeric golgi complex and its role in golgi traffic
Recently, 2 patients were identified to have a defect in subunit 7 of the conserved oligomeric Golgi complex (COG7); the patients were classified as CDG type IIe (74). The mammalian COG complex contains 8 subunits, of which COG1 through -4 form lobe A and COG5 through -8 form lobe B with COG4 as the core component linking the 2 lobes (75).
Mutations in COG subunits (COG1 through -8) of CHO, yeast, and Drosophila melanogaster sperm cells have been shown to affect the structure and function of the Golgi, producing defects in glycoconjugate biosynthesis, intracellular protein sorting, protein secretion, and in some cases, cell growth. In the recessive COG1- and COG2-null CHO mutants, for example, the Golgi showed an abnormal morphology with dilated cisternae and pleiotropic defects in several medial- and trans-Golgi–associated reactions affecting N-linked, O-linked, and lipid-linked glycoconjugates (76). The COG complex thus seems to play a role in determining and maintaining Golgi structure and morphology. Furthermore, COG works in concert with COPI. Their function is to retrograde transport several Golgi resident proteins to the appropriate Golgi compartment where they reside. Evidence came from the work of Oka et al. (77), who investigated the consequences of the loss and overexpression of COG on a set of Golgi resident type II transmembrane proteins, including members of the SNARE, Rab, and golgin protein families. The expression and localization of some proteins were COG-dependent, whereas for others this was not the case. The COG-sensitive proteins are referred to as "GEARs".
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Functions of O-Linked Glycans |
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TopAbstractIntroductionStructures of O-Linked GlycansBiosynthesis of O-GlycansFunctions of O-Linked GlycansCongenital Disorders in the...Screening Methods for Unraveling...References |
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Just like N-glycans, O-glycans can influence the secondary protein structure: the glycan can break the -helicity of peptides (80); can have a role in the tertiary protein structure [seen, e.g., on the porcine filamentous-shaped submaxillary mucin, in which release of an O-glycan leads to a globular shape (81)]; and in the quaternary protein structure and protein aggregation [seen, e.g., in ovine submaxillary mucin, which only forms aggregates when it is O-glycosylated (82)]. Subsequently, O-linked glycans maintain protein stability, heat resistance, hydrophilicity, and protease resistance by steric hindrance (8).
Additionally, mucin-type O-glycans are important for the binding of water. Mucins are proteins that are heavily glycosylated with mucin-type O-glycans and are often present at outer surfaces lacking an impermeable layer, such as the surfaces of the digestive, genital, and respiratory system tracts. These mucins bear clusters of sialylated glycans, which produce regions with a strong negative charge. This gives mucins the capacity to bind large amounts of water and form mucus. The gels observed in nasal secretions, for example, are formed by secreted MUC2 polypeptides linked together to form long, cross-linked polymers holding water. The primary function of these viscous mucin solutions and gels is to form a protective coating with antibacterial properties (83). Like mucin-type O-glycans, GAGs bind large volumes of water via the strong negative charge of the sulfate groups, providing resilience or resistance to compression rather than lubrication or reduction of friction. GAGs are found in extracellular matrices. In structural tissues, such as the cartilage of joints, GAGs can act as shock breakers by the slow reduction of their water content under high pressure.
Another important function of O-linked sugars is to mediate recognition between proteins. Glycan structures can be substrates for nonenzymatic sugar-binding proteins, known as lectins. By interacting with lectins, glycans influence the targeting of the proteins to which they are attached. Examples of glycan-mediated recognition of glycoproteins are ubiquitous. For example, selectins and galectins, representing 2 classes of lectins located in the leukocyte-vascular system, bind to carbohydrate epitopes that induce cellular signaling, which in turn influences many crucial cellular processes, including cell growth, apoptosis, endocytosis, cell-cell interactions, cell–matrix interaction, matrix network assembly, and oocyte fertilization (84). Additionally, sialylated O-mannosyl glycan serves as binding ligand for laminin in the dystroglycan complex, which is important in muscle and brain development (18). Moreover, O-linked glycans are known to have an effect on immunologic recognition; for example, the ABO blood group antigens and recognition of glycopeptides by the MHC complex or by antibodies (85).
Subsequently, it is known that GAGs have a role in nonspecific protein interactions. Cell surface proteoglycans, for example, adhere to soluble polypeptide growth factors through electrostatic interactions mediated by their GAGs, preventing the growth factors from diffusing. GAG interactions increase and stabilize concentration gradients of growth factors (78).
The effects of O-linked glycosylation on the bioactivity of many signaling molecules, particularly hormones and cytokines, and a relatively small number of enzymes, have been described. In most cases, the influence is not very strong (a difference of 2- or 3-fold), but rather provides a fine regulation mechanism. Effects leading to both an increase and a decrease in biological activity have been described. For example, a mucin-type O-linked glycan decreases the biological activity of interleukin-5 (86), whereas it induces a higher enzymatic activity of human lactase phlorizin hydrolase (87). The influence of unusual carbohydrate modifications on the activity of signaling molecules appears often to be crucial and specific. For example, O-Fuc on urinary-type plasminogen activator was shown to be required for activation of its receptor, and the presence of O-fucosyl glycans seems to be required for proper Notch function (22). Another example is the dynamic O-GlcNAc modification that seems to have an important role in a variety of signaling pathways, such as transcriptional regulation, proteasome-mediated protein degradation, insulin, and cellular stress signaling. Recently, it was found that O-GlcNAc modulates the activity of critical intermediates involved in the regulation of neutrophil motility (88). Some very specific GAG structures are known to act as co-receptors, allowing activation of the primary receptor necessary for the activation of growth factors. The fibroblast growth factor, for example, must interact with the heparan sulfate chain of the proteoglycan syndecan to activate the primary fibroblast growth factor receptor (89).
Finally, O-linked glycosylation is essential for the expression and processing of particular proteins. Glycophorin A, for example, is a heavily glycosylated protein present on the surface of human erythrocytes. It has been shown that O-linked sugars are necessary for cell surface expression of this glycoprotein (90). The influence of O-glycans in the processing of proteins is, for example, seen in pro-insulin-like growth factor II, which is cleaved into IGF-II only when Thr75 contains an O-linked sugar (91).
As O-glycans are involved in numerous processes, it is inevitable that defects in O-glycan biosynthesis might lead to severe abnormalities for cellular functioning.
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Congenital Disorders in the Biosynthesis of O-Glycans in Humans |
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TopAbstractIntroductionStructures of O-Linked GlycansBiosynthesis of O-GlycansFunctions of O-Linked GlycansCongenital Disorders in the...Screening Methods for Unraveling...References |
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This section discusses the clinical, molecular genetic, laboratory, and biochemical aspects of the known congenital disorders in the biosynthesis of O-glycans. The human congenital disorders that affect the biosynthesis of protein O-linked glycans are summarized in Table 3A
, whereas the human congenital disorders with a defect affecting the biosynthesis of both N- and O-glycans are summarized in Table 3B
. Both parts of Table 3
also list the group(s) who first discovered the genetic defects in the disorders.
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defects in mucin-type o-glycan biosynthesis
UDP-GalNAc transferase 3 (polypeptide N-acetylgalactosaminyltransferase 3) deficiency
The GALNT3 gene encodes UDP-GalNAc transferase 3 (GalNT3; EC 2.4.1.41), which transfers UDP-GalNAc to Thr/Ser of a protein backbone. GALNT3 is expressed in organs that contain secretory epithelial glands. It is highly expressed in human pancreas, skin, kidney, and testis and weakly expressed in prostate, ovary, intestine, and colon (92). Patients with familial tumoral calcinosis (FTC) can have mutations in the GALNT3 gene.
FTC. FTC is an autosomal recessive progressive metabolic disorder that manifests with massive calcium deposits in the skin and subcutaneous tissues and unresponsiveness to parathyroid hormone (93). At present, FTC is the only syndrome with an isolated defect in mucin-type O-glycan biosynthesis (94). The syndrome can be treated with phosphate-binding antacids (aluminum hydroxide) and a low-phosphorus diet combined with calcium deprivation, which reduces and prevents the recurrence of calcific masses (95).
defects in gag biosynthesis
ß-1,4-Galactosyltransferase 7 deficiency
The B4GALT7 gene encodes ß-1,4-galactosyltransferase 7 (B4GalT7; EC 2.4.1.133), which transfers Gal to the Xyl-Ser linkage in the linker region of proteoglycans (97). B4GALT7 is expressed in human heart, pancreas, liver, and to a lesser extent, in placenta, kidney, brain, skeletal muscle, and lung. Patients with the autosomal recessive progeroid variant of Ehlers–Danlos syndrome have mutations in the B4GALT7 gene.
Progeroid variant of Ehlers–Danlos syndrome. To date, 5 patients have been described with the progeroid variant of Ehlers–Danlos. Characteristic clinical features are a premature aging phenotype with a loose, elastic skin, failure to thrive, joint laxity, psychomotor retardation, hypotonia, and macrocephaly. Because proteoglycans are important structural components of the extracellular matrix of connective tissue, these patients suffer from skin, cartilage, and bone problems.
Deficiencies of extosin-1, -2, and -3
The EXT1 and EXT2 genes encode the proteins extosin-1 and -2, respectively, which oligomerize with the copolymerases (EC 2.4.1.224 and 2.4.1.225) responsible for the elongation of the heparin and heparan sulfate chains. The exact function of extosin-3 is not known. The EXT genes are ubiquitously expressed in human tissue. Patients with hereditary multiple exostoses (HME) have a mutated EXT1, EXT2, or EXT3 gene.
HME (types I, II, and III). HME is a genetically heterogeneous autosomal dominant disorder characterized by the development of multiple cartilage-capped benign bone tumors (exostoses) located mainly on the long bones. This disorder is often accompanied by skeletal deformities and short stature. In many cases, the exostoses transform to malignant tumors. Mutations in EXT1 and EXT2 account for 44%–66% and 30% of HME patients, respectively, whereas EXT3 appears to be the minor locus (100). For a review on hereditary multiple exostoses, please see the article by Wicklund et al. (101).
defects in gag sulfation
N-Acetylglucosamine-6-O-sulfotransferase deficiency
The CHST6 gene encodes human GlcNAc-6-O-sulfotransferase (EC 2.8.2.-) that transfers sulfate to the 6-O position of GlcNAc and Gal residues in the poly-N-acetyllactosamine extensions in keratan sulfate. GlcNAc-6-O-sulfotransferase is produced in human cornea, brain, spinal cord, and trachea. Macular corneal dystrophy (MCD) is caused by distinct mutations in the gene CHST6.
MCD (types I and II). MCD is a progressive autosomal recessive disease in which minute, gray, punctuate opacities in the cornea lead to bilateral loss of vision. Onset of clinical signs occurs in the first decade of life. Most patients have painful attacks with photophobia, foreign body sensations, and recurrent corneal erosions. MCD is characterized by nonsulfated (MCD type I) or low-sulfated (MCD type II) keratan sulfate (104).
Chondroitin 6-sulfotransferase 1 deficiency
The CHST3 gene encodes chondroitin 6-sulfotransferase 1 (EC 2.8.2.17), which catalyzes the sulfation of the 6-O position of GalNAc residues in chondroitin sulfate chains. CHST3 is widely expressed in adult tissues. It is expressed in the human heart, placenta, skeletal muscle, and pancreas, but also in various immune tissues such as the spleen, lymph nodes, and thymus. Recently, it was found that mutations in the CHST3 gene cause autosomal recessive spondyloepiphyseal dysplasia (SED), Omani type (111).
SED type Omani. Patients with SED Omani type are normal in length at birth but show growth retardation later, and are short in stature in adulthood (110–130 cm). Severe progressive kyphoscoliosis, severe arthritic changes with joint dislocations, rhizomelic limbs, genu valgum, cubitus valgus, mild brachydactyly, camptodactyly, and microdontia occur in this disease (112).
Diastrophic dysplasia sulfate transporter deficiency
The DTDST gene (also called SLC26A2) encodes the diastrophic dysplasia sulfate transporter (DTDST), which is a sulfate/chloride antiporter. The primary source of sulfur for the sulfation pathway of proteoglycans is free SO42–, which is transported to the cytoplasm mainly by DTDST. Mutations in the sulfate transporter lead to undersulfation of the GAGs. DTDST is ubiquitously expressed. Mutations in the DTDST gene are the cause of diastrophic dysplasia (DTD), achondrogenesis type 1B (ACGB1), atelosteogenesis type II (AO-II), and multiple epiphyseal dysplasia 4 (EDM4). The clinical features in the DTDST skeletal dysplasia family range from a relatively mild condition to severe conditions incompatible with life and are subdivided into the 4 syndromes listed above. The disorders have autosomal recessive inheritance. The severity of the phenotype correlates with the underlying DTDST mutation; mutations leading to stop codons or transmembrane domain substitutions mostly lead to the most severe phenotype (ACGB1), whereas other structural or regulatory mutations usually lead to one of the less severe phenotypes (113). The classification of DTD, AO-II, or EDM4, and thus of the severity of the disease, depends on residual sulfate uptake capacity and the extent of proteoglycan undersulfation (114). For a review, see the article by Rossi and Superti Furga (115).
ACGB1. ACGB1 is among the most severe skeletal disorders in humans. The disease is characterized by severe hypodysplasia of the spine, the rib cage, and the extremities. ACGB1 is always lethal immediately after and sometimes even before birth (113).
AO-II. AO-II is a lethal chondrodysplasia caused by collapse of the airways, resulting from abnormalities in the tracheal, laryncheal, and bronchial cartilage. Phenotypically, AO-II is the severe variant of DTD. In addition to the clinical features described for DTD, AO-II is characterized by severe and progressive kyphosis, horizontal sacrum, and a gap between the first and the second toes (116).
DTD. DTD is a skeletal dysplasia associated with short stature [adult height, 100–140 cm (117)], joint contractures, cleft palate, scoliosis, bilateral clubfeet, and characteristic clinical signs such as the so-called "hitchhiker thumb" and cystic swelling of external ears. Phenotypic variability is wide (115)(118).
EDM4. Patients with EDM4 have a condition with clubfoot, scoliosis, mild finger deformity, and mildly short or normal stature, but without palatal clefting, ear swelling, or thumb deviation (119).
3'-Phosphoadenosine 5'-phosphosulfate synthase 2 deficiency
The ATPSK2 gene encodes the enzyme 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2; EC 2.7.1.25/EC 2.7.7.4) (126). PAPSS2 is a bifunctional enzyme that activates cytoplasmic SO42– into a high-energy form in 2 enzymatic steps: (a) its ATP-sulfurylase uses ATP and SO42– to synthesize adenosine 5'-phosphosulfate (APS) and (b) its APS kinase catalyzes the phosphorylation of APS to 3'-phosphoadenosine 5'-phosphosulfate (PAPS). PAPS is the universal sulfate donor for posttranslational protein sulfation. Defective PAPSS2 thus leads to undersulfation of GAGs. PAPSS2 is produced in human cartilage. In a large Pakistani family, comprising 8 generations, a mutation was found in the ATPSK2 gene that leads to SED, Pakistany type.
SED, Pakistany type. The clinical features of SED, Pakistany type, include short stature evident at birth; short, bowed lower limbs; a mild, generalized brachydactyly, kyphoscoliosis, an abnormal gait, and early-onset degenerative joint disease in the hands and knees. Radiographs showed delayed epiphyseal ossification, especially of the hips and knees, and platyspondyly. Inheritance of the disease is autosomal recessive (127).
defects in o-galactosyl glycan biosynthesis
Lysyl hydroxylase-1 deficiency
The PLOD gene encodes lysyl hydroxylase-1 (EC 1.14.11.4). Lysyl hydroxylase-1 catalyzes the formation of hLys in collagens and other proteins with collagen-like amino acid sequences by the hydroxylation of Lys residues. hLys serves as attachment site for O-galactosyl glycans and is essential for the formations of collagen cross-links, contributing to collagen structure and stability. Lysyl hydroxylase-1 deficiency indirectly leads to an O-glycosylation defect. The function of the O-galactosyl glycans is unclear, although it is suggested that they may play a role in recognizing and activating collagen receptors in the cell membrane (129). Subsequently, it has been shown that there is a relationship between cross-link content and the degree of collagen glycosylation (130). Lysyl hydroxylase is produced in human liver, heart, lung, skeletal muscle, brain, and placenta (131). Patients with Ehlers–Danlos syndrome type VIa have mutations in the PLOD gene.
Ehlers–Danlos syndrome type VIa. Patients with Ehlers–Danlos type VIa are characterized clinically by neonatal kyphoscoliosis, generalized joint laxity, skin fragility, and severe muscle hypotonia at birth. Arterial rupture has caused death in some patients (132). The inheritance of the syndrome is autosomal recessive.
defects in o-mannosyl glycan biosynthesis
O-Mannosyl glycan biosynthesis disorders are characterized by an abnormal -dystroglycan glycosylation. -Dystroglycan is an essential component of the dystropin–glycoprotein complex, which is produced in human tissues such as muscle, brain, nerve, and heart. The dystrophin–glycoprotein complex is a multimeric transmembrane complex, providing a tight connection between the cytoskeleton and the extracellular matrix. Dystroglycan is generated from a single gene (DAG1) and is subsequently cleaved into 2 subunits: transmembrane ß-dystroglycan and peripheral -dystroglycan. In muscle, the intracellular side of transmembranic ß-dystroglycan binds to a variety of cytoplasmic molecules, such as dystropin, which in turn interacts with the cytoskeleton of cells. The extracellular side of ß-dystroglycan binds noncovalently to -dystroglycan, which in turn binds to extracellular matrix proteins such as laminin (135)(136). Different mammalian glycan sequencing studies have revealed that -dystroglycan is heavily glycosylated with O-linked Man chains (70%) and to a lesser extent with mucin-type O-glycans (30%), which mediate protein-protein interactions (137)(138). For the screening of defects in O-mannosyl glycan biosynthesis, the immunohistochemical staining of -dystroglycan is used on muscle biopsies of patients. At present, -dystroglycan is the only known substrate for this type of glycosylation in mammals. Often the monoclonal antibodies VIA4-1 and IIH6, which recognize an unknown carbohydrate epitope in -dystroglycan, are used. Antibodies against the core structures of - and ß-dystroglycan serve as controls in such experiments (139).
Protein O-mannosyltransferase deficiency
Coexpression of the POMT1 and the POMT2 genes is necessary for the enzymatic activity of protein O-mannosyltransferase [EC 2.4.1.109 (57)]. Mannosyltransferase catalyzes the attachment of Man residues to Thr/Ser amino acids of a protein. POMT1 is highly expressed in human testis, heart, and pancreas, whereas expression is lower in kidney, skeletal muscle, brain, placenta, lung, and liver. Walker–Warburg syndrome (WWS) and limb-girdle muscular dystrophy type 2K (LGMD2K) can be caused by mutations in the POMT1 gene. POMT2 is highly expressed in testis, with expression lower in most tissues. Recently, it was discovered that mutations in the POMT2 gene also cause WWS (140).
WWS. In 20% of WWS patients, a mutation is found in the POMT1 gene. The incidence of POMT2 mutations is in the same range as that of POMT1(140). The phenotype seen in the WWS patients with a POMT2 mutation is indistinguishable from that of patients with POMT1 mutations. Patients with this rare autosomal recessive disorder have a life expectancy of <3 years (mean, 0.8 years). WWS patients have malformations of the muscle, eye, and brain. Typical brain anomalies include hydrocephalus, cerebellar hypoplasia, absent corpus callosum and cerebellar vermis, cobblestone cortex, and fusion of the hemispheres. Additionally, WWS patients can have numerous eye anomalies, such as cataracts, microphthalmia, persistent hyperplastic primary vitreous, and Peters anomaly. WWS patients have little motor activity because of severe muscle dystrophy (141). For a review, see van Reeuwijk et al. (141).
LGMD2K. Patients with LGMD2K have progressive muscle weakness involving the proximal muscles of the shoulder and pelvic girdles. These patients also have a slow, progressive limb-girdle muscular dystrophy, a mild microcephaly, and severe mental retardation, but normal brain imaging. Onset of the autosomal recessive disorder is in the first decade of life (142)(143).
-dystroglycan by the VIA4-1 and/or IIH6 monoclonal antibodies was abnormal (142)(144)(145). Decreased staining of the laminin 2-chain (merosin) has been observed in WWS, although patients have been reported with normal amounts of merosin(146)(147).
O-Mannosyl-ß1,2-N-acetylglucosaminyltransferase-1 deficiency
The POMGNT1 gene encodes the enzyme O-mannosyl-ß1,2-N-acetylglucosaminyltransferase-1 (EC 2.4.1.101). This enzyme catalyzes the second step, the linkage of a GlcNAc residue to protein-bound Man, in the O-mannosyl glycan core structure. The enzyme O-mannosyl-ß1,2-N-acetylglucosaminyltransferase-1 appears to be present in all tissues. Muscle–eye–brain disease (MEB) is caused mainly by mutations in the POMGNT1 gene.
MEB. MEB is a muscular dystrophy/neuronal migration disorder with a phenotype similar to, but less severe than, that of WWS patients. The life expectancy of MEB patients is 10–30 years (144). Clinically, MEB is differentiated from WWS mainly on the basis of the presence of a normal or thin corpus callosum and of pronounced cerebellar cysts, which are both absent in WWS patients (144). The inheritance of the disorder is autosomal recessive. For a review, see Diesen et al. (149).
-dystroglycan by VIA4-1 and/or IIH6 monoclona
