Carbohydrate-De¢Cient Glycoprotein Syndrome Type II

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Biochimica et Biophysica Acta 1455 (1999) 179^192 www.elsevier.com/locate/bba

Review Carbohydrate-de¢cient glycoprotein syndrome type II

Harry Schachter a;*, Jaak Jaeken b

a Department of Biochemistry, University of Toronto Medical School, and Department of Structural Biology and Biochemistry, Hospital for Sick Children, 555 University Avenue, Toronto, Ont. M5G 1X8, Canada b Center for Metabolic Disease, University of Leuven, Leuven, Belgium

Received 8 February 1999; accepted 3 May 1999

Abstract

The carbohydrate-deficient glycoprotein syndromes (CDGS) are a group of autosomal recessive multisystemic diseases characterized by defective glycosylation of N-glycans. This review describes recent findings on two patients with CDGS type II. In contrast to CDGS type I, the type II patients show a more severe psychomotor retardation, no peripheral neuropathy and a normal cerebellum. The CDGS type II serum transferrin isoelectric focusing pattern shows a large amount (95%) of disialotransferrin in which each of the two glycosylation sites is occupied by a truncated monosialo-monoantennary N-glycan. Fine structure analysis of this glycan suggested a defect in the Golgi enzyme UDP-GlcNAc:K-6-D-mannoside L-1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) which catalyzes an essential step in the biosynthetic pathway leading from hybrid to complex N-glycans. GnT II activity is reduced by over 98% in fibroblast and mononuclear cell extracts from the CDGS type II patients. Direct sequencing of the GnT II coding region from the two patients identified two point mutations in the catalytic domain of GnT II, S290F (TCC to TTC) and H262R (CAC to CGC). Either of these mutations inactivates the enzyme and probably also causes reduced expression. The CDG syndromes and other congenital defects in glycan synthesis as well as studies of null mutations in the mouse provide strong evidence that the glycan moieties of glycoproteins play essential roles in the normal development and physiology of mammals and probably of all multi- cellular organisms. ß 1999 Elsevier Science B.V. All rights reserved.

Keywords: Carbohydrate-de¢cient glycoprotein; N-Acetylglucosaminyltransferase; N-Glycan synthesis; Golgi; Transferrin glycoform; Psychomotor retardation

Contents

1. Introduction ...... 180

2. Clinical presentations of CDGS ...... 180

3. The analysis of serum transferrin glycoforms in CDGS ...... 181

4. Clinical biochemistry ...... 182

5. Complex N-glycan synthesis within the Golgi apparatus ...... 183

* Corresponding author.

BBADIS 61859 15-9-99 180 H. Schachter, J. Jaeken / Biochimica et Biophysica Acta 1455 (1999) 179^192

6. The genetic defect in CDGS II ...... 184

7. The diagnosis of CDGS II ...... 186

8. The function of complex N-glycans in development ...... 186

Acknowledgements ...... 187

References ...... 187

1. Introduction transferrin pattern have been described (designated types Ia, Ib and Ic). The enzymatic and genetic de- At least 0.5^1% of the transcribed human genome fects in these variants have been determined and it is is devoted to the production of proteins involved in clear that there are other CDGS type I patients with the synthesis, degradation and function of glycocon- as yet undetermined metabolic defects. CDGS types jugates [1]. It is therefore not surprising that there III [5] and IV [6] cases are very rare and nothing is are many congenital diseases with defects in these known about the biochemical defects in these pa- pathways. Table 1 lists eight autosomal recessive dis- tients. eases in which there is a defect in the synthesis of Asn-linked glycans (N-glycans) proving that these structures are essential for normal human develop- 2. Clinical presentations of CDGS ment. Some of these diseases are discussed in other chapters of this volume. This chapter will focus on CDGS I occurs world-wide and a¡ects both sexes carbohydrate-de¢cient glycoprotein syndrome type II equally [4,7]. The patients show moderate to severe (CDGS II). neurological disease (cerebellar ataxia and marked The carbohydrate-de¢cient glycoprotein syn- psychomotor retardation), a characteristic dysmor- dromes are a group of congenital multi-systemic dis- phy and variable involvement of other organs. Feed- eases characterized by defective glycosylation of N- ing problems, vomiting and diarrhea may occur re- glycans. The discovery of this disease was based on sulting in severe developmental delay and failure to the observation by Jaeken et al. [2] of decreased se- thrive [8,9]. Infants may show a distinctive lipodys- rum thyroxine-binding globulin and increased aryl- trophy (peculiar distribution of subcutaneous fat), sulfatase A activity in two patients with familial psy- nipple retraction and hypogonadism. After infancy, chomotor retardation. In 1984, Jaeken et al. [3] retinitis pigmentosa [10,11], joint contractures, skel- reported sialic acid de¢ciency in serum and cerebro- etal deformities [12], stroke-like episodes, epilepsy spinal £uid transferrin from identical twin-sisters and peripheral neuropathy may develop. About with demyelinating disease and thereby ¢rst showed 20% of the patients die within the ¢rst year. Lan- that this new syndrome was due to defective protein guage and motor development are severely delayed glycosylation. There were about 280 patients with and walking without support is rarely achieved. The this disease world-wide as of October, 1998 [4]. Until IQ ranges from 40 to 60 and the children usually recently, four variants of CDGS were recognized have an extroverted and cheerful disposition. Com- (called types I^IV) based primarily on the quantita- puterized tomography (CT), nuclear magnetic reso- tive analysis of serum transferrin glycoforms either nance imaging (MRI) and postmortem studies of the by isoelectric focusing or chromatofocusing. Varia- brain of CDGS I patients often show olivopontocer- tions in the pattern of transferrin glycoforms re£ect ebellar atrophy (OPCA) and a small brain stem [13^ di¡erences in negative charge due to abnormal sialyl- 20]. ation of protein-bound N-glycans (described in more Two CDGS patients, designated as CDGS II, have detail below). Within the last 3 years, at least three been described who di¡er from the classic picture of distinct variants of CDGS associated with a type I CDGS I described above, an Iranian girl (patient A)

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[21] and a Belgian boy (patient B) [22,23]. In contrast 1^7%; S3, 4^17%; S4, 46^73%; S5, 10^30%; S6, 0^ to CDGS I, both CDGS II patients had a more 9%; S7, traces. The corresponding values for CDGS severe psychomotor retardation, no peripheral neu- sera showing a type 1 pattern [3,20,31,33,36^38] are: ropathy and a normal cerebellum on MRI. Both pa- S2, 14^37%; S3, 6^19%; S4, 18^57%; S5, 5^17%; S6, tients had a similar dysmorphy particulary of the 0^6%. In addition, CDGS I sera show appreciable face (coarse features, low-set ears), widely spaced amounts of S0 (2^29%), a glycoform not usually de- nipples, a ventricular septal defect, a striking stereo- tected in normal sera. In other words, CDGS I sera typic behavior (tongue thrusting, hand washing and show dramatic reductions in S4 and S5 and dramatic other movements), generalized hypotonia and limb increases in S0 and S2 (Fig. 1). Data from many weakness, and normal deep tendon re£exes. Patient laboratories indicate an `all-or-none' situation in A was lost from follow-up after the age of 3 years. which each glycosylation site on transferrin is either Patient B's speech was limited to a few monotonous occupied by a normal N-glycan or is not occupied sounds at 10 years. He su¡ered from frequent infec- suggesting that the addition of oligosaccharide to tions in infancy and epilepsy developed at 6 years. Asn is the defective step (Fig. 1) [13,35,39^42]. The diagnosis of CDGS was not made until patient The CDGS II serum transferrin isoelectric focus- B was 9.5 years old when an investigation of bleeding ing pattern di¡ers markedly from that in CDGS I gums revealed a typical CDGS coagulopathy. Radio- (Fig. 1) in that there are no or minimal amounts of logical examination showed osteopenia and other asialo-, tetrasialo-, pentasialo- and hexasialo-trans- skeletal abnormalities. MRI revealed absence of cer- ferrrins, low amounts of monosialo-transferrin, a ebellar atrophy, but showed delayed myelination and moderate amount (about 4%) of trisialo-transferrin global cortical atrophy. and a large amount (95%) of disialo-transferrin [23,28,29]. Electrospray-mass spectrometry analysis of both 3. The analysis of serum transferrin glycoforms in normal and CDGS II puri¢ed serum transferrin CDGS showed seven glycoform peaks (I^VII) with molecular masses ranging from 79 265 to 80 369 Da for nor- The basic defect in CDGS is defective N-glycosyl- mal transferrin and 77 958 to 79 130 Da for CDGS II ation of glycoproteins throughout the body, which transferrin [28]. On the basis of a polypeptide chain explains the severe multi-systemic nature of the dis- molecular mass of 75 143 Da, it was calculated that ease. Diagnosis can be made by examination of the the major transferrin species III (79 557 and 78 247 complex N-glycans on any glycoprotein. The most Da for normal and CDGS II transferrin, respec- convenient source of glycoproteins is serum and the tively) are, respectively, tetrasialo-transferrin (S4) in protein usually used is transferrin because of the rel- which each glycosylation site is occupied by a disialo- ative simplicity of its glycosylation. Serum transferrin biantennary N-acetyllactosamine type N-glycan and has only two asparagine N-glycosylation sites per disialo-transferrin (S2) in which each glycosylation molecule, both of which are normally occupied by site is occupied by a truncated monosialo-monoan- complex N-glycans, primarily of the disialo-bianten- tennary N-glycan (Fig. 1). The minor peaks I, V and nary N-acetyllactosamine type with smaller amounts VI for normal transferrin represent respectively S3 of monosialo-biantennary, disialo-triantennary, tri- (disialo-biantennary and monosialo-biantennary N- sialo-triantennary and tetrasialo-tetraantennary N- glycans), S4 (disialo-biantennary and disialo-trian- glycans (Fig. 1) [13,24^30]. Normal serum transferrin tennary) and S5 (disialo-biantennary and trisialo-tri- therefore exists as a series of glycoforms containing antennary). The corresponding peaks for CDGS II two to seven sialic acid residues per mole of protein transferrin represent respectively S1 (truncated (designated S2 to S7, Fig. 1). Chromatofocusing, iso- monosialo-monoantennary and asialo-monoanten- electric focusing, capillary zone electrophoresis and nary N-glycans), S2 (truncated monosialo-mono- anion exchange chromatography have been used to antennary and monosialo-biantennary) and S3 analyze human serum transferrin glycoforms [3,31^ (truncated monosialo-monoantennary and disialo- 35]. The normal glycoform concentrations are: S2, biantennary) (Fig. 1). Transferrin species I and V

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Fig. 1. Structures of S0^S7 transferrin glycoforms. Serum transferrin has two asparagine N-glycosylation sites per molecule. In normal transferrin (structures in upper section of ¢gure), both sites are occupied by complex N-glycans, primarily of the disialo-biantennary N-acetyllactosamine type with smaller amounts of monosialo-biantennary, disialo-triantennary, trisialo-triantennary and tetrasialo-tetraantennary N-glycans. Normal serum transferrin exists as a series of glycoforms containing two to seven sialic acid residues per mol of protein (designated S2^S7). Box I shows the abnormal S0^S3 underglycosylated structures found in CDGS I (the `all-or-none' structures, see text). Box II shows the abnormal S1^S3 underglycosylated structures found in CDGS II. The major glycoform in normal transferrin is the S4 structure marked with an X. The major glycoforms in CDGS I are: (1) the normal S4 structure marked with an X; (2) the two S2 structures marked with an X in Box I; and (3) the S0 structure in Box I. The major glycoform in CDGS II is the abnormal S2 structure marked with an X in Box II. The insert in the lower right of the ¢gure shows a diagram of the isoelectric focusing pattern of the major serum transferrins in normal controls (C), CDGS I (I) and CDGS II (II). (References to serum transferrin analysis: [3,13,24^41].) a, GlcNAc; F, Man; b, Gal; E, sialic acid. correspond to the desialylated forms of species III The large amount of normal transferrin glycoform and VI, respectively. Peaks II and IV are probably S4 in CDGS I serum indicates that this mutation is sulfated derivatives and peak VII has not been iden- quite leaky. In contrast, the mass spectrometry data ti¢ed. [28] indicate that there is little, if any, normal S4 in CDGS II serum transferrin was isolated by immu- CDGS II serum. The small amount of S4 seen on noa¤nity chromatography and separated into minor isoelectric focusing is probably a truncated derivative and major fractions by fast protein liquid chroma- of S6 (Fig. 1). Thus the mutation in CDGS II ap- tography. The structure of the glycans released from pears to be stringent. the major fraction by hydrazinolysis was established by application of methanolysis and high resolution proton NMR spectroscopy [28]. The major N-glycan 4. Clinical biochemistry is the truncated monosialo-monoantennary N-glycan ManK1^6[NeuNAcK2^6GalL1^4GlcNAcL1^2Man- Since there is defective glycosylation of all N-gly- K1^3]Man-R where R is -L1-4GlcNAcL1-4GlcNAc- cosylated glycoproteins, it is not surprising that Asn-X. The presence of truncated N-glycans clearly many glycoproteins in CDGS I serum (transporters, di¡erentiates the CDGS II transferrin glycoform pat- hormones, complement factors, clotting factors and tern from the `all-or-none' model described above for inhibitors, lysosomal enzymes and others) [2,3,20,36^ CDGS I (Fig. 1). The parents of the CDGS II pa- 38,43^55], cerebrospinal £uid [3,56], leukocytes [50], tients showed a normal pattern. ¢broblasts [57,58] and liver [37] show abnormal het-

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erogeneity due to incomplete glycosylation. Not only dase, K1-antitrypsin, apolipoprotein B, haptoglobin). is there hypoglycosylation of serum glycoproteins but Serum IgM and cerebrospinal £uid protein were ele- the levels of various serum glycoproteins may be de- vated. creased or increased probably secondary to damage Human L-trace protein is a major intrathecally to the liver and other organs or to missorting, defec- synthesized polypeptide constituent of human cere- tive reuptake and/or reduced stability of underglyco- brospinal £uid (CSF). The L-trace protein from the sylated glycoproteins, e.g. glycoprotein transport two CDGS II patients showed a single di-N-glycosy- along the secretory pathway in CDGS I skin ¢bro- lated protein band with a signi¢cantly lower molec- blasts may be delayed and dilation of the rough en- ular weight than the di-N-glycosylated polypeptide doplasmic reticulum has been observed indicating re- from control CSF [56]. The major oligosaccharide tention of selected glycoproteins [59]. structures of the glycoprotein from CDGS II patients Although many of the abnormal serum glycopro- were asialo-monoantennary and monosialo-monoan- tein levels are similar for both CDGS I and II, there tennary lactosamine-type chains with a core fucose are some signi¢cant di¡erences, e.g. CDGS II pa- and a bisecting GlcNAc. tients show absence of proteinuria, a de¢ciency of clotting factors IX and XII, decreased L-glucuroni- dase, and normal serum glutamic pyruvic transami- 5. Complex N-glycan synthesis within the Golgi nase activity, albumin, cholesterol and arylsulfatase apparatus A. As occurs in CDGS I, patient B showed decreased levels of many serum glycoproteins (thyroxine-bind- The CDGS type I serum transferrin glycoform ing globulin, clotting factor XI, antithrombin III, pattern implies a leaky mutation in either oligosac- proteins C and S, heparin cofactor II, L-galactosi- charyltransferase (OT, Fig. 2) or in one of the steps

Fig. 2. Metabolic pathway from glucose and mannose to dolichol pyrophosphate oligosaccharide. Enzymes: HK, hexokinase; GK, glucokinase; PGI, phosphoglucoisomerase; PGM, phosphoglucomutase; PMI, phosphomannoisomerase; PMM, phosphomannomutase; Glc-T, glucosyltransferase; Man-T, mannosyltransferase; oligo-T, oligosaccharyltransferase. Asterisks on PMM, PMI and Glc-T denote enzyme defects in CDGS Types Ia, Ib and Ic, respectively (Table 1). Intermediates: G-6-P, glucose-6-phosphate; G-1-P, glucose-1-phosphate; F-6-P, fructose-6-phosphate; M-6-P, mannose-6-phosphate; M-1-P, mannose-1-phosphate; UDPG, UDP-glucose; Dol-P-Glc, dolichol phosphate glucose; Dol-P-Man, dolichol phosphate mannose; dolichol-PP-(GlcNAc)2, dolichol pyrophosphate N-acetylchitobiose; dolichol-PP-oligosaccharide, dolichol pyrophosphate (Glc)3(Man)9(GlcNAc)2. Dolichol pyrophosphate (Glc)3(Man)9(GlcNAc)2 is assembled by the addition of Man from both GDP-Man and Dol-P-Man and Glc from Dol-P-Glc to dolichol-PP-(GlcNAc)2.

BBADIS 61859 15-9-99 184 H. Schachter, J. Jaeken / Biochimica et Biophysica Acta 1455 (1999) 179^192 in the synthetic pathway to dolichol pyrophosphate is the ¢rst committed step in the conversion of hybrid oligosaccharide (Fig. 2). These mutations occur in to complex N-glycans (Fig. 3) [61,65^68]. GnT I and the rough endoplasmic reticulum or cytoplasm MII are type II integral membrane glycoproteins fac- (Figs. 2 and 3). Leukocyte adhesion de¢ciency type ing the lumen of the Golgi membrane [69]. At least II and galactosemia (Table 1) are also cytoplasmic some patients with HEMPAS have a de¢ciency of diseases. However, inclusion cell disease, CDGS II MII [70,71]. and HEMPAS (Table 1) are diseases in which there UDP-GlcNAc:K-6-D-mannoside L-1,2-N-acetylglu- is a defect in the processing of N-glycans within the cosaminyltransferase II (GnT II; EC 2.4.1.143) cata- Golgi apparatus (Fig. 3). The ¢rst committed step in lyzes incorporation of a GlcNAc residue in L1,2 link- the conversion of oligomannose to complex and hy- age to the ManK1^6 arm of ManK1^6[GlcNAcL1^ brid N-glycans is catalyzed by UDP-GlcNAc:K-3-D- 2ManK1^3]ManL1-R to form GlcNAcL1^2ManK1^ mannoside L-1,2-N-acetylglucosaminyltransferase I 6[GlcNAcL1^2ManK1^3]ManL1-R and is therefore (GnT I; EC 2.4.1.101, Fig. 3). GnT I adds a GlcNAc an essential step in the biosynthetic pathway leading residue to the ManK1^3ManL1-R arm of ManK1^ from hybrid to complex N-glycans (Fig. 3). GnT II is 6(ManK1^3)ManK1^6[ManK1^3]ManL1-R to form a typical type II transmembrane protein [64,72] and ManK1^6(ManK1^3)ManK1^6[GlcNAcL1^2ManK1^ has been mapped to human chromosome 14q21. 3]ManL1-R where R is -4GlcNAcL1-4GlcNAc-Asn- X. GnT I action is an essential pre-requisite for the action of several enzymes leading to complex N-gly- 6. The genetic defect in CDGS II cans, K-3/6-mannosidase II, GlcNAc-transferases II, III and IV and core K-1,6-fucosyltransferase [60,61]. The truncated N-glycans found in CDGS II serum The human GnT I gene (MGAT1) has been mapped transferrin (Fig. 1) indicate that CDGS II patients to chromosome 5q35 [62^64]. have a defect in GnT II. Indeed, GnT II activity Processing of the product of GnT I, ManK1^ was reduced by over 98% in ¢broblast extracts 6(ManK1^3)ManK1^6[GlcNAcL1^2ManK1^3]Man- from the two unrelated CDGS II patients [23] and L1-R, by K-3/6-mannosidase II (MII, EC 3.2.1.114) there was no detectable GnT II activity in mononu- to form ManK1^6[GlcNAcL1^2ManK1^3]ManL1-R clear cell extracts from patient B (Fig. 4) [73].

Table 1 Autosomal recessive disorders with defective N-glycan synthesis Name of disease Type of disease Enzyme defect Reference Inclusion cell disease Lysosomal glycoprotein storage Phospho-N-acetyl glucosaminyl-transferase required [112^116] (I-cell disease or disease with severe developmental for synthesis of Man-6-phosphate targeting signal (mucolipidosis II) abnormalities Leukocyte adhesion Immunode¢ciency and severe Defective synthesis of GDP-fucose [117^120] de¢ciency type II developmental abnormalities (LAD II) Carbohydrate-de¢cient glycoprotein syndromes (CDGS) CDGS type Ia Severe developmental abnormalities Phosphomanno-mutase 2 defect (85% of Type 1) See text and [4,20,121,122] CDGS type 1b Gastrointestinal^hepatic problems Phosphomannose isomerase defect [123^125] CDGS type 1c Developmental abnormalities Glucosyltransferase defect [86,126] (also called type V) CDGS type II Severe developmental abnormalities GlcNAc-transferase II de¢ciency See text and [21^23,73,74] HEMPAS (congenital Relatively mild disease with mild K-Mannosidase II de¢ciency. Other factors? See text and dyserythropoietic anemia and hemosiderosis [127^129] anemia type II) Galactosemia Failure to thrive, liver disease, mental (i) Galactokinase; (ii) galactose-1-phosphate [79^81] retardation, cataracts uridyltransferase

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Fig. 3. Pathway from dolichol pyrophosphate oligosaccharide to complex N-glycans. Oligosaccharyltransferase (OT) transfers

(Glc)3(Man)9(GlcNAc)2 from dolichol pyrophosphate oligosaccharide to an Asn residue in a nascent peptide chain in the rough endoplasmic reticulum. The glycosylated peptide is released from the ribosome into the lumen and undergoes processing by K-glucosidases and K-mannosidases as it moves through the lumen of the endoplasmic reticulum and Golgi apparatus. In the medial Golgi compart- ment, the successive actions of N-acetylglucosaminyltransferase I (GnT I), K-mannosidase II (MII) and N-acetylglucosaminyltransferase II (GnT II) convert the oligomannose N-glycan (Man)5(GlcNAc)2-peptide to the biantennary complex N-glycan (GlcNAc)2(Man)3(GlcNAc)2-peptide. The defects in CDGS Types Ia, Ib and Ic (Table 1, Fig. 2) all result in defective synthesis of dolichol pyrophosphate oligosaccharide which therefore results in defective OT action. HEMPAS and CDGS II are due, respectively, to defects in the Golgi enzymes MII (at least in some cases) and GnT II.

Direct sequencing in both directions of the entire GnT II coding region from the two patients identi- ¢ed two point mutations in the catalytic domain of GnT II, S290F (TCC to TTC) in patient A and H262R (CAC to CGC) in patient B [74]. Both mutations occur in the C-terminal catalytic domain [61,75] at locations which are conserved between rat and human GnT II [64]. Both patients are homozygous for their respective mutations and have therefore inherited the same allele from each parent. The father, mother and brother of patient B show one normal allele and one allele with the same mutation as patient B. The CDGS II mutations were introduced into the normal MGAT2 gene followed by expression in the baculovirus/Sf9 system [74]. No enzyme activity was detected in cells transfected by either of the two mu- Fig. 4. GnT II analyses on mononuclear cell extracts (from patient B, his blood relatives and controls) expressed as a ratio of tant genes (less than 1% of the control value). West- activity of GnT II relative to activity of GnT I [29,73]. The as- ern blot analysis showed that both mutant proteins signment of genotype is based on restriction endonuclease anal- were expressed at about 8% of the level of normal ysis of DNA for the presence of the GnT II gene mutation GnT II expression indicating that the mutations ei- [74]. Symbols: encircled solid square, CDGS II patient B; solid ther interfere with transcription^translation or lower squares, related heterozygotes; solid circles, blood relatives of patient B who are homozygous normals; open squares, controls the stability of the protein in the baculovirus/Sf9 sys- unrelated to patient B. Mononuclear cell extracts from all 13 tem. Northern blot analysis showed normal tran- heterozygotes have GnT II levels which are 32^67% lower than scription of the GnT II gene in CDGS II ¢broblasts. normal levels.

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It remains to be established whether there is a defect is not possible by this method [84]. Abnormal glyco- of either translation or protein stability in human form patterns in CDGS I appear in the second to cells. The data suggest that the mutations reduce third weeks of post-natal life. It is now possible to protein expression and also inactivate GnT II en- carry out pre-natal testing of CDGS II by GnT II zyme activity. It is of interest that although the mu- assays and mutation analysis of the GnT II gene tations in the two patients are di¡erent, they cause using chorionic villus samples, cultured chorionic vil- the identical phenotype. lus cells or cultured amniocytes [85]. A thorough Restriction endonuclease analysis of DNA from 23 molecular work-up of both the patient and family blood relatives of one of these patients showed that should probably be carried out on all future sus- 13 donors were heterozygotes; the other relatives and pected cases of CDGS. 21 unrelated donors were normal homozygotes [74]. Screening for CDGS transferrin glycoforms by iso- All heterozygotes showed a signi¢cant reduction (33^ electric focusing or an equivalent technique is recom- 68%) in mononuclear cell GnT II activity (Fig. 4). mended for patients not only with psychomotor re- Analysis of patient B's family proves that CDGS II tardation and hypotonia, but also in unexplained is a recessive autosomal disease located at chromo- feeding problems with failure to thrive, liver ¢brosis, some 14q21. decreased coagulation factor XI and antithrombin III, and intestinal disease, even in the absence of severe neurological disease. Although very useful, 7. The diagnosis of CDGS II the analysis of glycans on serum transferrin glycoforms can be misleading since hypoglycosylation of The characteristic serum transferrin glycoform pat- glycosyltransferases and glycosidases may a¡ect their tern (Fig. 1) is the most commonly used diagnostic activity or intracellular localization, thereby produc- criterion for CDGS. Iron saturation is required for ing secondary alterations in carbohydrate structure accurate analysis of transferrin glycoforms and only [86]. serum or heparin plasma should be used [76,77]. It is essential to perform a detailed glycoform analysis for the diagnosis of the various CDGS types since car- 8. The function of complex N-glycans in development bohydrate-de¢cient serum transferrin also occurs in other conditions, such as chronic alcohol intake [78], The synthesis of complex N-glycans is divided into galactosemia [79^81], untreated fructosemia [82,83] three distinct stages (Figs. 2 and 3). The ¢rst stage and hepatic disease [78]. occurs primarily in the cytoplasm and rough endo- Once a diagnosis of CDGS II has been con¢rmed plasmic reticulum and involves the synthesis of doli- by demonstration of the typical transferrin (or other chol pyrophosphate Glc3Man9GlcNAc2. The second glycoprotein) glycoform pattern (Fig. 1), GnT II as- stage begins with the transfer of Glc3Man9GlcNAc2 says should be carried out on fresh monocytes (Fig. oligosaccharide to an Asn residue and involves proc- 4) or on cultured ¢broblast or lymphoblast extracts. essing to Man5GlcNAc2-Asn-X, the substrate for Diagnosis can also be made by GnT II assays on GnT I. The third stage completes complex N-glycan parental monocytes (Fig. 4). The diagnosis can be synthesis and occurs primarily in the Golgi appara- con¢rmed by molecular mass determination of serum tus. transferrin with electrospray-mass spectrometry Complex N-glycans are absent from bacteria [87] [28,39^41]. The diagnosis should be completed by and yeast [88] and are present in very small amounts, mutational analysis [74], although this may not be if at all, in protozoa (Trypanosoma cruzi [89], Leish- a simple matter due to the strong possibility that mania [90] and Plasmodium [91]) and Dictyostelium unrelated cases of CDGS II will have di¡erent muta- discoideum [92]. However, all of the above organisms tions. except bacteria are capable of making N-glycans of The serum glycoprotein glycoform patterns are the oligomannose type and therefore express the en- normal in the CDGS I fetus, and probably also in zymes of stage one and at least some of the enzymes the CDGS II fetus, and therefore pre-natal diagnosis of stage two of N-glycan synthesis.

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Complex N-glycans are present in most of the mul- growth of cells in tissue culture, they play critical ti-cellular invertebrate and vertebrate animals that roles in cell^cell interactions and the morphogenesis have been analyzed, i.e., nematodes [93^95], schisto- of multi-cellular organisms. somes [96^98], molluscs [99,100], insects [101], ¢sh In conclusion, the CDG syndromes and other con- [102], birds [103^106] and mammals. These species genital defects in glycan synthesis provide strong evi- therefore express all three stages of N-glycan synthe- dence that the glycan moieties of glycoproteins play sis. essential roles in the normal development and phys- Tunicamycin is an antibiotic which inhibits the iology of mammals and probably of all multi-cellular ¢rst enzyme in the synthesis of dolichol pyrophos- organisms. phate Glc3Man9GlcNAc2 and therefore prevents all N-glycosylation of proteins [107]. Tunicamycin inhibits replication in yeast, fungi, protozoa, enveloped Acknowledgements viruses and mammalian cell lines in culture [107]. Yeast temperature-sensitive mutants with defects in These studies were supported by a Medical Re- the steps leading to the Man5GlcNAc2-Asn-X core search Council of Canada grant to H.S. and by a are not viable at the restrictive temperature, whereas Nationaal Fonds voor Wetenschappelijk Onderzoek mutations in later stages of oligosaccharide assembly of Belgium grant to J.J. are not lethal [88,108]. Children with CDGS types 1a and 1c with defects in the ¢rst stage of N-glycan synthesis show moderate to severe psychomotor retardation and other multi-systemic abnormalities. References Therefore stage one and at least part of stage two of N-glycan synthesis are highly conserved in evolu- [1] A. Varki, J. Marth, Oligosaccharides in vertebrate development, Semin. Dev. Biol. 6 (1995) 127^138. tion and are essential for organisms from yeast to [2] J. Jaeken, M. Vanderschueren-Lodeweyckx, P. Casaer, L. man. It is not known why oligomannose N-glycans Snoeck, L. Corbeel, E. Eggermont, R. Eeckels, Familial psy- are essential for the viability of uni-cellular organ- chomotor retardation with markedly £uctuating serum pro- isms. lactin, FSH and GH levels, partial TBG de¢ciency, increased The third stage of N-glycan synthesis and complex serum arylsulphatase A and increased CSF protein: a new N-glycans probably appeared in evolution just prior syndrome?, Pediatr. Res. 14 (1980) 179. [3] J. Jaeken, H.G. van Eijk, C. van der Heul, L. Corbeel, R. to the development of multi-cellular organisms sug- Eeckels, E. Eggermont, Sialic acid-de¢cient serum and cere- gesting that complex N-glycans are required for the brospinal £uid transferrin in a newly recognized genetic syn- cell^cell interaction process and normal development drome, Clin. Chim. Acta 144 (1984) 245^247. of multi-cellular animals. In support of this concept, [4] H. Carchon, E. Van Schaftingen, G. Matthijs, J. Jaeken, it has been shown that somatic Chinese hamster ova- Carbohydrate-de¢cient glycoconjugate syndrome Type IA (phosphomannomutase de¢ciency), Biochim. Biophys. ry cell mutants lacking the GnT I gene show essen- Acta, in press. tially normal growth in tissue culture, whereas mouse [5] H. Stibler, B. Westerberg, F. Hanefeld, B. Hagberg, Carbo- embryos with a null mutation in this gene do not hydrate de¢cient glycoprotein (CDG) syndrome ^ a new survive beyond 10.5 days post-fertilization and variant, type III, Neuropediatrics 24 (1993) 51^52. show severe developmental abnormalities particu- [6] H. Stibler, U. Stephani, U. Kutsch, Carbohydrate-de¢cient larly of the brain [109,110]. Mice with a homozygous glycoprotein syndrome ^ a fourth subtype, Neuropediatrics 26 (1995) 235^237. null mutation in the gene encoding GnT II survive to [7] H.H. Freeze, M. Aebi, Molecular basis of carbohydrate-de- term, but are born stunted with various congenital ¢cient glycoprotein syndromes Type I with normal phospho- abnormalities and die shortly after birth (J. Marth, mannomutase activity, Biochim. Biophys. Acta, in press. personal communication and [111]). Children with [8] M.B. Petersen, K. Brostrom, H. Stibler, F. Skovby, Early CDGS type II have a defective GnT II gene and manifestations of the carbohydrate-de¢cient glycoprotein syndrome, J. Pediatr. 122 (1993) 66^70. show severe psychomotor retardation and other mul- [9] B. Kristiansson, S. Borulf, N. Conradi, C. ErlansonAlberts- ti-systemic abnormalities. These studies indicate that son, W. Ryd, H. Stibler, Intestinal, pancreatic and hepatic although complex N-glycans are not essential for the involvement in carbohydrate-de¢cient glycoprotein syn-

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