Primary contributions to this chapter were made by J.B. Lowe (HHMI/University of Michigan, Ann Arbor) and H. Freeze (The Burnham Institute, La Jolla, California).

THIS CHAPTER DEALS WITH INHERITED DISEASES in glycan biosynthesis. It focuses on an emerging group of carbohydrate-deficient glycoprotein syndromes (CDGS), leukocyte adhesion deficiency syndrome II (LADII), congenital dyserythropoietic anemia type II (HEMPAS), galactosemia, and abnormalities in proteoglycan synthesis. For a discussion of inherited deficiencies in the blood group glycosyltransferases, see Chapters 16 and 17. Chapter 18 discusses lysosomal storage diseases resulting from defects in glycan degradation, and Chapter 23 discusses disorders of lysosomal enzyme phosphorylation. Diseases such as cystic fibrosis where altered glycosylation is a secondary effect are covered in Chapter 37.

Introduction

Inherited disorders of glycosylation are biochemically and clinically heterogeneous. Individuals with CDGS, LADII, HEMPAS, and galactosemia typically suffer from malfunction of multiple organ systems. Depending on the specific biosynthetic lesion, the structures and function of many glycoproteins, glycolipids, and glycophosphotidylinositol-linked proteins may be affected. Recent remarkable progress in this area has led to the identification of several primary genetic defects and to successful therapies based on clear biochemical rationales. In addition, these studies imply that many of the diseases are probably more genetically heterogeneous than once expected and are probably underdiagnosed. Consequently, a new awareness of this group of disorders by the clinical community should foster new insights into their pathogenesis and treatment.

Spontaneous Mutations in Humans and Animal Models

Most of this chapter focuses on human glycosylation disorders; however, animals also show spontaneous mutations in glycosylation. The defects in animals primarily affect proteoglycan biosynthesis, since these defects produce dysmorphic features that are easy to recognize. Table 32.1 lists disorders in animals and humans (see similar tables in Chapters 7 and 33). In addition, Chapter 23 covers mutations in several animal species for many of the lysosomal storage disorders.

Table 32.1

Naturally occurring disorders in glycosylation pathways in animals.

Clinical and Laboratory Features of the CDGSs ^(1–10)

The CDGSs are an emerging group of clinically heterogeneous autosomal recessive glycosylation disorders. Four types of CDGSs (types I through IV) have been defined on the basis of their serum transferrin isoelectric focusing profile (see below). Systematic CDGS nomenclature is still being refined and will probably be revised in the near future. CDGS type I is the most common, with more than 200 patients described in the medical literature. In contrast, only a few patients with CDGS types II, III, and IV have been reported. CDGS type I has now been subdivided into three distinct forms, termed CDGS type Ia, CDGS type Ib, and CDGS type Ic (also called type V), according to the nature of the clinical symptoms and molecular defects. Types II, III, and IV are single distinct clinical entities at this time.

The diagnosis of CDGS is based on clinical signs and symptoms, together with isoelectric focusing or chromatofocusing analysis of serum transferrin. These methods provide a very sensitive indicator of the glycosylation state of serum transferrin. Other serum glycoproteins have altered glycosylation, but transferrin is the most reliable, sensitive, and simplest indicator.

In normal humans, serum transferrin molecules have two glycosylation sites that are occupied primarily (~80%) by disialylated, biantennary N-glycans. Most molecules contain four sialic acid residues, and they exhibit a characteristic electrophoretic migration position (Figure 32.1). This tetrasialylated transferrin glycoform is termed S4. Approximately 10–15% of normal human serum transferrin molecules contain at least one trisialylated, triantennary N-glycan providing S5 and S6 glycoforms. These forms increase during an acute-phase response but are not important for CDGS diagnostics. In CDGS patients, the S4 glycoform is either substantially diminished (CDGS type I) or absent (CDGS type II), with compensatory increases in two other glycoforms, termed S2 and S0. Structural analyses of the S2 and S0 glycoforms isolated from CDGS type I patients show that each S2 transferrin molecule contains only a single disialylated biantennary N-glycan (at one of the two normal attachment sites), whereas each S0 molecule is completely deficient in N-glycan modification (Figure 32.1). At present, mutations in three different genetic loci account for CDGS underglycosylation. In CDGS type II, the glycans are efficiently added, but they are processed abnormally. In type IV, structurally altered glycans are added from the lipid precursor and are also abnormally processed.

Figure 32.1

Schematic diagram of the glycan structures found on serum transferrin in normal individuals and on serum transferrin in CDGS types I and II. The approximate fraction of total serum transferrin that exhibits a particular glycoform is indicated at the right. (more...)

CDGS Type Ia ^(11–25)

CDGS type Ia is the most common form of CDGS, representing approximately 70% of all cases of CDGS type I. More than 180 patients with this disease have been reported in the literature since its discovery in 1980. These individuals typically present at birth with hypotonia, dysmorphic features, failure to thrive, liver dysfunction, and a pronounced susceptibility to infection. Approximately 20% of these patients die within the first few years of life. Other clinical features of CDGS type Ia include hypoplasia of the cerebrum, cerebellum, and brainstem, and a significant delay in motor and language development. CDGS patients also exhibit an unusual distribution of body fat, and suffer from polyneuropathy, ataxia, hypotonia, skeletal deformities, retinitis pigmentosa, and oculomotor abnormalities. Liver function abnormalities are characterized by elevated serum transaminases, decreased serum albumin, and clinically significant decrements in the blood-clotting proteins antithrombin III, protein C, protein S, heparin cofactor, and factor XI. CDGS type Ia patients also exhibit abnormalities in endocrine homeostasis and typically display hypogonadism.

Biochemical and molecular genetic analyses disclose that the glycosylation defect in CDGS type Ia is caused by defects in the phosphomannomutase 2 locus (PMM2). This gene is on human chromosome 16p13, where linkage studies have localized the CDGS type I locus in some families. The coding region of the PMM2 locus shares approximately 65% DNA sequence identity with the PMM1 locus. PMM1 is found on human chromosome 22q13, which excludes the involvement of this gene in the classical form of CDGS type Ia. A processed PMM2 pseudogene, termed PMM2psi, localizes to human chromosome 18p.

The PMM2 locus encodes an enzyme that catalyzes an essential step in the biosynthesis of GDP-Man and Dol-P-Man (Figure 32.2). These two compounds are essential substrates for the mannosyltransferases required for the synthesis of the lipid-linked precursor for N-glycosylation (Figure 32.2). In CDGS type Ia, a deficiency of phosphomannomutase activity reduces the levels of GDP-Man and Dol-P-Man. The reduced levels of these compounds reduce synthesis of Glc₃Man₉GlcNAc₂-PP-Dol, the physiological substrate for oligosaccharyltransferase. Reductions in GDP-Man and Dol-P-Man also yield truncated forms of the Dol-PP-oligosaccharide. However, these truncated forms, and especially those that are not glucosylated, are poor substrates for oligosaccharyltransferase. Oligosaccharyltransferase preferentially uses the physiological precursor substrate Glc₃Man₉GlcNAc₂-PP-Dol, but its low level is rate-limiting, causing the enzyme to leave some asparagine-linked sites unmodified. Nevertheless, a substantial proportion of sites receive a normal Glc₃Man₉GlcNAc₂ glycan that is trimmed and remodeled normally, to give a disialylated biantennary oligosaccharide.

Figure 32.2

Synthetic reactions in N-glycan biosynthesis that are affected in CDGS. The reactions affected in CDGS types Ia, Ib, and Ic, and type IV are indicated by the parenthetic annotation below the relevant synthetic step.

It is important to point out that GDP-Man is also required for the synthesis of the four mannose residues of glycophosphotidylinositol anchors (see Chapter 10) and is the major source of GDP-Fuc required for terminal fucosylation (see Leukocyte Adhesion Deficiency II Syndrome below). The diminished synthesis of GDP-Man in PMM-deficient CDGS type Ia is therefore likely to cause deficits in the structure and function of molecules that depend on these processes. For example, it is possible that the frequent and severe infectious complications suffered by CDGS type Ia patients may be due to defective synthesis of leukocyte selectin ligands, which require terminal α1–3-fucosylation for proper function (see Chapter 26, and Leukocyte Adhesion Deficiency II Syndrome below).

The sequence of the PMM2 locus has been examined in more than 50 CDGS patients with phosphomannomutase deficiency. A large fraction of these patients are compound heterozygotes for missense mutations in the PMM2 locus. Less commonly, CDGS type Ia patients are homozygous for missense mutations. Some PMM-deficient patients do not have mutations in the coding region of the PMM2 gene. These latter observations suggest that PMM deficiency can also be caused by mutations that affect the regulation of this gene or the splicing and stability of its transcripts. Nevertheless, existing and emerging information about the nature of PMM mutations in PMM deficiency will help advance encouraging early efforts to establish prenatal diagnosis in CDGS type Ia.

Therapeutic approaches to CDGS type Ia remain problematic. In vitro studies demonstrate that addition of exogenous mannose to CDGS type Ia fibroblasts can diminish the synthesis of short lipid-linked oligosaccharides observed in these cells and is associated with an increase in the incorporation of radioactive mannose into glycoproteins. These results suggested the possibility that mannose supplementation might ameliorate the defect in CDGS type Ia patients. However, the biochemical basis for these interesting and encouraging in vitro results is not yet clear, and mannose therapy in CDGS type Ia patients has not been effective at the biochemical level, nor does it seem to improve the clinical status of CDGS type Ia patients.

CDGS Type Ib ^(26–29)

CDGS type Ib is a recently described form of CDGS. There are about ten known patients, but several have died before diagnosis. Patients with CDGS type Ib do not present with psychomotor or developmental abnormalities. Instead, these individuals present in the first year of life with hypoglycemia, severe vomiting and diarrhea, protein-losing enteropathy, and hepatic fibrosis. CDGS type Ib patients are also susceptible to recurrent thromboses, presumably due to their low levels of antithrombin III. The isoelectric-focusing pattern of serum transferrin in CDGS type Ib is identical to that observed in type Ia. Phosphomannomutase activity is normal in CDGS type Ib; however, phosphomannose isomerase activity is deficient in these patients (Figure 32.2). Molecular analyses identified missense mutations in the phosphomannose isomerase (PMI) locus of CDGS type Ib patients. As in CDGS type Ia, PMI deficiency (Figure 32.2) is expected to lead to decreased levels of GDP-Man, Dol-P-Man, and Glc₃Man₉GlcNAc₂-PP-Dol and elaboration of truncated forms of this precursor. Again, this circumstance leads to the synthesis of glycoproteins that are incompletely glycosylated with structurally correct N-glycans. In principle, these patients may also have defects in the synthesis of other fucosylated glycans and some glycophosphotidylinositol linkages.

A therapeutic approach to bypass PMI deficiency in CDGS type Ib was suggested by the position of PMI within the metabolic pathway leading to GDP-Man and Dol-P-Man (Figure 32.2). Under normal circumstances, Man-6-P can be derived both from Fru-6-P, via PMI, and from free mannose, via hexokinase. This latter pathway remains intact in CDGS type Ib patients. Mannose in this pathway presumably comes from glycoconjugates degraded in the lysosome and from the blood where it is delivered by one or more mannose-specific transporters. Circulating mannose presumably derives from dietary sources, including mannose-rich glycans in plant and animal foodstuffs. Mannose in these glycans is liberated from the complex carbohydrates in food through α-mannosidases that are located in the brush border of intestinal enterocytes. Apparently, the amounts of mannose derived from a normal diet, and from glycoprotein catabolism, are insufficient to circumvent the PMI defect in CDGS type Ib via the hexokinase-dependent pathway. However, it is conceivable that the flux of mannose through this pathway could yield enough Man-6-P, and its downstream products, to spare CDGS type Ib patients from the more severe clinical signs and symptoms characteristic of CDGS type Ia.

These considerations prompted efforts to circumvent the PMI defect by providing a supraphysiological flux of mannose through the hexokinase route to Man-6-P synthesis. In vitro studies with PMI-deficient cells confirm that extracellular mannose can be used by CDGS type Ib cells to fully compensate for PMI deficiency. In addition, in vivo studies demonstrate that oral administration of mannose elevates serum mannose levels well above the K_uptake of the mannose transporter. These observations provided the scientific rationale for initiating chronic oral mannose therapy for several PMI-deficient CDGS type Ib patients. This approach was quite successful and corrected hypoglycemia, protein-losing enteropathy, and intermittent gastrointestinal problems. It increased plasma antithrombin III levels into the normal range, and corrected the isoelectric-focusing pattern of serum transferrin and other serum glycoproteins. Since orally administered mannose is well-tolerated, this approach is clearly a satisfyingly effective, if not curative, therapy for this life-threatening condition.

CDGS Type Ic ^(30,31)

CDGS type Ic (also called type V) has a transferrin glycoform phenotype identical to that observed in CDGS types Ia and Ib, but PMM and PMI are both normal. CDGS type Ic was identified quite recently. Two families have been reported in the literature, but nine more have also been identified. The patients described in published reports have a mild CDGS type Ia phenotype, which includes psychomotor abnormalities, involving diminished mentation capacity, and deficiency of motor development. The patients also suffer from recurrent infections and have decreased levels of several blood-clotting factors.

Biochemical analyses of type Ic fibroblasts show deficient synthesis of Glc₃Man₉GlcNAc₂-PP-Dol, and an accumulation of Man₉GlcNAc₂-PP-Dol, which is a poor substrate for oligosaccharyltransferase (Figure 32.2). Dol-P-Glc and UDP-Glc syntheses are normal, but the glucosyltransferase activity that adds the first glucosyl residue to the Man₉GlcNAc₂-PP-Dol precursor is virtually undetectable in these fibroblasts by in vitro assays. The marked reduction in glucosyltransferase activity results from mutations in the glucosyltransferase-coding sequence. Unfortunately, understanding the biochemical lesion in these patients does not provide an obvious therapeutic approach. Palliative treatment is all that can be offered at this time to these patients.

It is important to point out that the defect in CDGS type Ic is confined to the N-glycosylation pathway, since cells remain competent to synthesize normal levels of Man-6-P, GDP-Man, and Dol-P-Man. This may account for the relatively milder clinical presentation compared to that of CDGS type Ia patients, where defective synthesis of Man-6-P could affect N-glycosylation, GPI anchor synthesis, and fucosylation, since GDP-Man is converted to GDP-Fuc.

CDGS Type II ^(32–36)

Two families have been described with CDGS type II. The patients have dysmorphic features and psychomotor retardation that is even more severe than that typically seen in CDGS type Ia. In addition, CDGS type II patients have a ventricular septal defect, but they do not have peripheral neuropathy or cerebellar atrophy. They are relatively deficient in the blood-clotting factors XI, antithrombin III, protein C, protein S, and heparin cofactor, as are other CDGS patients, but they are also deficient in factors IX and XII. CDGS type II is easily distinguished from types Ia, Ib, and Ic by transferrin isofocusing (see Figure 32.1). In CDGS type II, the tetrasialylated glycoform S4 is virtually absent and is replaced by the disialylated glycoform S2. Both glycosylation sites are occupied by truncated asparagine-linked glycans (Figure 32.3) missing the “second” antenna which is normally initiated by the enzyme N-acetylglucosaminyltransferase II (GlcNAcT-II; see Chapter 7). Biochemical analyses demonstrated approximately 98% reduction in GlcNAcT-II activity in CDGS type II fibroblasts. Molecular analyses show that the patients are homozygous for enzyme-inactivating missense mutations in the GlcNAcT-II locus. There is no effective therapy for this disorder at present.

Figure 32.3

N-glycans that accumulate in CDGS type II and in HEMPAS. (Normal) N-glycans that are present on red cell bands 3 and 4.5; “n” may be from 1 to several, and represents the number of lactosamine units within the polylactosamine chain. CDGSII (more...)

CDGS Types III and IV ^(37–41)

CDGS type III has been described in two families. This disorder is characterized by severe stationary psychomotor retardation with tetraparesis, cerebral and optic atrophy, and dysmorphic features. The clinical symptoms differ from CDGS type I in that polyneuropathy, retinal pigmentation, and cerebellar hypoplasia are absent. Serum transferrin glycoform analysis shows a predominance of the tetrasialylated isoform S4 but a somewhat increased level of all the hyposialylated glycoforms.

CDGS type IV has been reported in two families. This form of CDGS is characterized by psychomotor retardation associated with microcephaly and epileptic seizures and by an apparent absence of liver disease. Serum transferrin glycoform analysis shows a relative excess of the disialylated transferrin glycoform. One patient with this transferrin pattern and these clinical features is defective in Dol-P-Man synthase activity. The patient primarily makes a truncated lipid-linked oligosaccharide with only five mannose residues, compared to the normal individual with nine residues. Although this oligosaccharide is transferred to proteins normally, subsequent oligosaccharide processing in fibroblasts is abnormal. Hybrid chains with only one sialic acid occur more frequently. This alteration probably accounts for the increase in S2 transferrin seen in this patient. Addition of mannose to the culture medium of fibroblasts corrects the truncated size of the precursor oligosaccharide and altered processing. The reason for the correction is uncertain, but the K_m for Dol-P-Man synthase is about fivefold higher than normal in the patient. Presumably, increasing exogenous mannose increases the local concentration of GDP-Man pool and drives the reactions. It is important to stress that a deficiency in Dol-P-Man will also affect synthesis of glycophospholipid anchors, C-mannosylated proteins, and possibly the O-mannose-based glycans seen in brain.

There are other recent reports of infants with neonatal psychomotor abnormalities, and other clinical abnormalities, associated with aberrant serum transferrin glycoforms. Nearly all of these patients were identified through transferrin analysis because they shared a few symptoms with patients having known forms of CDGS. Transferrin is a powerful diagnostic tool because it can theoretically detect the consequences of mutations in at least 30 glycosylation-related genes. It remains to be seen whether such alterations actually have clinical consequences. Transferrin is a useful marker, but it cannot be used to detect other altered glycosylations such as polylactosamines or fucosylation, since the N-glycans of transferrin do not contain these structures.

Leukocyte Adhesion Deficiency II Syndrome ^(42–53)

The term leukocyte adhesion deficiency (LAD) was originally chosen to describe a syndrome characterized by frequent, nonpurulent bacterial infections, a leukocyte adhesion defect, and peripheral blood leukocytosis. There now appear to be two types of these deficiencies, termed LADI and LADII. The molecular basis of LADI has been shown to be due to diminished or absent expression of the integrin-type leukocyte adhesion molecules LFA-1 (CD11a/CD18; α_Lβ₂), Mac-1 (CD11b/CD18; α_Mβ₂), and p150/95 (CD11c/CD18; α_Xβ₂), or normal expression of dysfunctional forms of these molecules. Mutations in the gene encoding the β-subunit (CD18) common to these heterodimeric β₂ integrins account for the pathogenesis of this disease. The clinical and laboratory findings are all compatible with the integrin-dependent leukocyte adhesion defect.

More recently, a LAD-like syndrome has been described in two unrelated boys with normal levels and function of CD18. Like LADI, there are recurrent, nonpyogenic infection and leukocytosis. However, in addition to the CD18-dependent symptoms of LADI, these patients also present with decreased growth rate, severe mental retardation and other neurological manifestations, and various morphological and skeletal abnormalities not observed in LADI patients. Furthermore, both patients exhibit the rare Bombay blood group phenotype, characterized by a deficiency in red cell H blood group structures (see Chapter 16). Both patients are also deficient in the Lewis blood group antigens and are non-Secretors of the blood group antigens. Because the H and Lewis antigens correspond to fucosylated oligosaccharide structures, and because the non-Secretor trait is also associated with an absence of expression of fucosylated blood group substance (see Chapter 16), these observations suggested that these patients might have a general defect in fucose metabolism. Since fucosylated leukocyte antigens, such as sialyl Lewis X, contribute to selectin-dependent leukocyte adhesion (discussed in Chapter 28), defective fucose metabolism could also account for the LAD-like phenotype in these patients. In fact, their leukocytes do not express sialyl Lewis X. These observations prompted Etzioni and colleagues to call β₂-integrin-dependent disease LAD type I (LADI) and the sialyl Lewis X deficiency as LAD type II (LADII).

Subsequent studies showed that LADII neutrophils lack ligands for E-selectin, P-selectin, and L-selectin. This is implied by flow cytometry studies showing the absence of binding the monoclonal antibody CSLEX-1, a surrogate marker for E- and P-selectin ligand activity. LADII neutrophils do not adhere to E-selectin or P-selectin in vitro, nor do such cells participate in L-selectin-dependent homotypic aggregation events in vitro. In vivo, intravital microscopy studies demonstrate that LADII neutrophils do not roll on venular endothelium under circumstances where endothelial E-selectin is expressed and mediates leukocyte rolling under shear flow. Finally, skin window and skin chamber tests in a LADII patient document a profound reduction in migration of neutrophils and monocytes into inflamed cutaneous sites. Considered together, these observations demonstrate that LADII leukocytes do not maintain normal trafficking due to a deficiency in selectin ligands. These observations, and the leukocytosis characteristic of the LADII syndrome, are consistent with the defective leukocyte-trafficking phenotypes. They are also consistent with the leukocytosis observed in P-selectin and/or E-selectin null mice and in mice deficient in FucT-VII, the α1–3 FucT that makes an essential contribution to leukocyte selectin ligand activity.

As noted above, LADII patients are deficient in three different fucosylated blood group antigens (the H/ABO, Secretor, and Lewis systems), and in the fucosylated leukocyte antigen recognized by the CSLEX-1 antibody. In principle, this constellation of deficiencies could be accounted for by homozygous null alleles at each of the four fucosyltransferase loci making these antigens (the H, Se, Lewis, and FucT-VII loci). However, it is now clear that the defect instead lies in the pathway involving the synthesis of GDP-Fuc (Figure 32.4).

Figure 32.4

Synthesis of GDP-Fuc. The cytosolic de novo pathway begins with GDP-Man and is catalyzed by two enzymes (a 4,6-dehydratase, and the FX protein) that yield GDP-Fuc. Alternatively, the “salvage” pathway begins with cytosolic fucose derived (more...)

De novo biosynthesis of GDP-Fuc involves three separate reactions that occur in the cytosol of mammalian cells (see Chapter 6). This pathway begins with the conversion of GDP-Man to GDP-4-keto-6-deoxymannose by the enzyme GDP-Man-4,6-dehydratase. GDP-4-keto-6-deoxymannose is then converted to GDP-Fuc via a two-step reaction involving a 3,5-epimerase-dependent conversion to GDP-4-keto-6-deoxy-l-Gal, and a subsequent 4-reductase-dependent formation of GDP-Fuc (Figure 32.4). In mammalian cells, a single polypeptide catalyzes the epimerization and reduction reactions. This epimerase-reductase polypeptide is also known as the FX protein. The FX protein was first described as a “tumor-specific” antigen and was also discovered as an abundant red cell protein with an unknown function. Molecular cloning studies revealed that the FX protein shares primary sequence similarity with prokaryotic epimerase-reductase enzymes involved in GDP-Fuc synthesis. Biochemical studies have confirmed that the FX protein functions as the GDP-4-keto-6-deoxymannose 3,5-epimerase-4-reductase.

Mammalian cells can also synthesize GDP-Fuc using the salvage or “scavenger” pathway. Extracellular fucose can be transported into the cytosolic compartment via fucose-specific plasma membrane transporters. Alternatively, fucose cleaved from endocytosed glycoproteins can enter the cytosol. Fucose kinase forms Fuc-1-P, and GDP-Fuc pyrophosphorylase (GDP-Fuc “synthase”) makes GDP-Fuc from Fuc-1-P. Cell culture experiments suggest that the scavenger pathway makes a relatively minor contribution to the GDP-Fuc pools.

Following its synthesis in the cytosol, GDP-Fuc is subsequently transported into the lumen of the Golgi apparatus (see Chapter 6). Luminal-localized GDP-Fuc serves as a substrate for Golgi-localized fucosyltransferases that fucosylate membrane-associated and soluble glycoconjugates.

Studies exploring GDP-Fuc metabolism in cultured cells derived from LADII patients indicate that the defect in this disorder lies in the de novo pathway of GDP-Fuc biosynthesis. Under normal culture conditions, Epstein-Barr-virus-transformed B lymphocyte cell lines prepared from LADII patients do not bind the fucose-specific lectin Lotus tetragonobolus agglutinin, confirming that LADII cells do not express fucosylated cell surface glycans. However, growth of the LADII cells in fucose-containing media restores lectin binding. These observations demonstrate that the salvage pathway for GDP-Fuc synthesis is intact in LADII cells and imply that transport of GDP-Fuc into the Golgi lumen is not impaired (Figure 32.4). In contrast, in vitro studies reveal that the conversion of GDP-Man to the GDP-4-keto-6-deoxymannose intermediate is defective, whereas the epimerase-reductase reaction is apparently unaffected by the LADII lesion. However, 4,6-dehydratase activity exists in an inactive form in LADII cytosol but becomes active after a preincubation step. Furthermore, although the 4,6-dehydratase reaction is impaired in LADII cells, the coding sequence of the LADII dehydratase locus is intact, and the 4,6-dehydratase protein is expressed at normal levels in LADII cells. These observations imply that the defect in LADII involves an unknown factor that may control the de novo GDP-Fuc synthetic pathway, via an interaction with the GDP-Man4,6-dehydratase.

A recent report indicates that the immunological impairment in LADII may diminish during maturation, since there seems to be a reduction in the incidence of infectious episodes in these patients as they grow older. Since the fucosylation defect in LADII cells can be corrected via the salvage pathway, a fucose-supplemented diet might lead to clinical improvement, since it might partially restore defective leukocyte selectin ligand activity. This potential therapy is not without risk, since it might also restore H blood group determinant expression in red cells, as well as A and/or B determinants, depending on the patient's ABO genotype. Because LADII patients, like most individuals with the Bombay phenotype, maintain high titers of the IgM class, complement fixing anti-A, anti-B, and anti-H antibodies, robust restoration of these red cell antigens by oral fucose therapy could precipitate an episode of acute, autoimmune hemolytic anemia.

Despite the possible complications, fucose supplementation was cautiously tested on the most recently diagnosed LADII patient. This 2-year-old boy presented with all of the typical LADII symptoms. The primary defect was not identified, but no mutations were found in the coding sequences of either the dehydratase or the reductase-epimerase, and the respective mRNA levels were normal. Fibroblasts from the patient were defective in fucosylation, but it was restored by providing exogenous fucose. On the basis of this finding, the patient was given multiple daily doses of fucose, which were efficiently absorbed into the blood. Soon after beginning therapy, some fucosylated selectin ligands appeared on neutrophils and core fucosylation of serum glycoproteins returned. During 5 months of treatment, infections and fever disappeared, elevated neutrophil counts returned to normal, and, surprisingly, psychomotor capabilities even improved based on standardized tests. Fortunately, H-antigen did not appear on the red cell surface and there was no hemolytic anemia. These results underscore the ability of the salvage pathway to contribute directly to fucosylation. The variable effectiveness of fucose therapy in restoring fucosylation suggests that phenotypic correction may be cell-type-specific and glycoconjugate-selective. Very little is known about fucose utilization or how it is regulated in higher animals (see Chapter 6)

Congenital Dyserythropoietic Anemia Type II ^(54–64)

Hereditary erythroblastic multinuclearity with a positive acidified-serum lysis test (HEMPAS) is also known as congenital dyserythropoietic anemia type II. It appears to be an autosomal recessive disorder, but it is not clear if the primary defect is in glycosylation (see Chapter 37). The acronym HEMPAS refers to the observation that serum from some normal individuals contains an antibody that binds to red cells from HEMPAS patients and will lyse these cells when the serum is acidified. The nature of the antigen recognized by this antibody in not known, nor is it understood why this antibody is present in some but not all normal human sera. In HEMPAS patients, the red cells and their marrow precursors are fragile and susceptible to lysis, which accounts at least in part for the ineffective production of red cells by the marrow. Hyperplasia of the erythroid precursors in the marrow is observed in HEMPAS patients and is accompanied by the presence of multinucleated erythroblasts. Ultrastructural analyses disclose abnormal membrane structure in cells of the erythroid lineage. Patients with HEMPAS suffer from the consequences of ineffective erythropoiesis, including anemia, marrow hyperplasia, enlarged spleen, gallstones, and liver disease, with excessive accumulation of iron in the liver. The disease primarily affects the marrow red cell progenitors (erythroblasts) and the red cells themselves, but the biochemical defects discussed below have sometimes been found in leukocytes and other tissues in some individuals.

Analysis of red cell glycans in HEMPAS discloses a structural defect in N-glycans borne by a pair of red cell membrane proteins termed band 3 (the anion exchange protein) and band 4.5 (the glucose transporter protein). These proteins normally bear biantennary complex N-glycans that are decorated with long polylactosamine on both antennae. However, in HEMPAS patients, red cell bands 3 and 4.5 are virtually devoid of polylactosamine. In contrast, these cells contain relatively large amounts of polylactosamine on glycolipids (i-antigen), which are relatively minor components in normal red cells.

Structural analyses of the band-3 and band-4.5 glycans indicate that these vary to some significant extent among different individuals with HEMPAS. In some such patients, the abnormal glycans feature a truncation of the antenna attached to the α1–6-linked mannose residue of the trimannosyl core structure in the N-glycan (Figure 32.3). This structure is the immediate synthetic precursor to GlcNAcT-II, and its excessive accumulation has implied a defect in the expression or activity of this enzyme in some HEMPAS patients (Figure 32.4). Although it has been reported that the cells in some HEMPAS patients exhibit 70–90% reduction in GlcNAcT-II activity, the pathophysiological relevance of these observations is not clear, since the inherited GlcNAcT-II deficiency that causes CDGS type II is clearly associated with a much more severe disease and involves multiple organ systems. It has been suggested that a red-cell-specific decrement in GlcNAcT-II could account for the erythroid-specific manifestations in HEMPAS, versus the pan-lineage deficit of this enzyme characteristic of CDGS type II. However, it is not clear how an erythroid-specific deficiency of GlcNAcT-II could cause the virtually complete deficit of band-3 polylactosamine observed in HEMPAS red cells, especially since these glycans are only reduced by approximately 50% in CDGS type II. These considerations make it unlikely that simple defects in the GlcNAcT-II locus will be found to account for HEMPAS.

In other HEMPAS patients, hybrid-type N-glycans predominate and are characterized by the retention of the α1–3- and α1–6-linked mannose residues attached, in turn, to the α1–6-linked mannose of the trimannosyl core (Figure 32.3). This structure is the synthetic substrate for α-mannosidase II and must be processed by this latter enzyme before GlcNAcT-II may act. The excessive accumulation of this partially processed glycan in the red cells of some HEMPAS patients has implied a defect in expression or activity of α-mannosidase II in these individuals (Figure 32.3). In one such patient, α-mannosidase II activity is virtually absent in some cells, and decreased accumulation of the α-mannosidase II RNA transcript seems to account for the enzyme deficiency in this family. However, the molecular defect is unknown, but it may lie in another locus that regulates its expression.

Why is the apparent α-mannosidase II deficiency in these HEMPAS cases limited to the erythroid lineage? From studies of mice rendered deficient in α-mannosidase II, the best explanation is the presence of another α-mannosidase II-like activity (termed α-mannosidase III) in extra-erythroid tissues. Additionally, recent molecular cloning studies have identified a related α-mannosidase locus, termed α-mannosidase IIx. The gene that encodes α-mannosidase III activity has not yet been identified.

In summary, the molecular basis for HEMPAS is not clearly understood. Genetic linkage studies in some HEMPAS pedigrees have excluded defects in the α-mannosidase II locus, the α-mannosidase IIx locus, and the GlcNAcT-II locus. Furthermore, the biochemical basis for diversion of erythroid polylactosamine biosynthesis from N-glycans to glycolipids is not at all clear. These observations, together with evidence for significant genetic heterogeneity implied by glycan structural analyses, indicate that significant challenges lie ahead in understanding the pathophysiology of this disorder.

Galactosemia ^(65–70)

Galactosemia refers to a group of diseases caused by inherited defects in the genes encoding three enzymes in galactose metabolism. One of these disorders, termed classical galactosemia, is caused by a deficiency of Gal-1-P uridyl transferase (GALT; Figure 32.5). This disease may decrease synthesis and availability of UDP-Gal. Defects in UDP-Gal 4′ epimerase or in galactokinase are rare, do not apparently impact on UDP-Gal synthesis or accumulation, and are not discussed further.

Figure 32.5

UDP-Gal synthesis and galactosemia. The most common form of galactosemia is due to a deficiency of Gal-1-P uridyltransferase (GALT). This enzyme normally utilizes Gal-1-P derived from dietary galactose. In the absence of GALT, Gal-1-P accumulates, along (more...)

GALT-deficient individuals present in infancy with a failure to thrive, enlarged liver, jaundice, and cataracts. Institution of a lactose-free diet ameliorates most of the acute symptoms of the disorder. This treatment reduces the amount of galactose entering the galactose metabolic pathway and thereby diminishes the accumulation of excessive amounts of galactose and Gal-1-P that are thought to contribute to the symptoms of the disease. The reduction in galactose accumulation also helps to inhibit the formation of galactitol and galactonate, which are produced via reductive or oxidative metabolism of galactose, respectively. Galactitol is not metabolized further and has osmotic properties that can make a dominant contribution to cataract formation. Unfortunately, a galactose-free diet apparently does not prevent the appearance of cognitive disability, ataxia, growth retardation, and ovarian dysfunction characteristic of this disease. It has been suggested that these long-term complications in treated GALT-deficient individuals may be due to small amounts of toxic metabolites that continue to accumulate in these patients (via small amounts of dietary galactose and via de novo synthesis from Glc-1-P; Figure 32.5). It has also been suggested that GALT deficiency leads to a relative deficit of UDP-Gal, the nucleotide sugar substrate used by galactosyltransferases, with a consequent deficiency of galactosylated glycans that may contribute to the pathogenesis of this disease. Hypogalactosylation of glycoproteins and glycolipids has been observed in some GALT-deficient individuals and seems to support this hypothesis. In addition, patients not adhering to galactose-free diets synthesize abnormal transferrin glycoforms typical of CDGS types 1a and 1b. The pattern returns to normal when they return to galactose-free diets. However, it is not clear whether GALT-deficient patients have a physiologically relevant deficit of cellular UDP-Gal, especially since significant amounts of this compound are formed from UDP-Glc by UDP-Gal 4′ epimerase (Figure 32.5). It is possible that accumulation of hypogalactosylated glycans is secondary to a general metabolic abnormality in these patients. A relationship between the accumulation of these abnormal structures and the neurological deficits in these patients remains to be demonstrated.

Defects in Proteoglycan Synthesis ^(71–79)

Proteoglycans and their glycosaminoglycan GAG chains are critical components in extracellular matricies. For a discussion of their biosynthesis, core proteins, and function, see Chapter 11.

Ehlers-Danlos syndrome (progeroid type) is a connective tissue disorder characterized by failure to thrive, loose skin, skeletal abnormalities, hypotonia, and hypermobile joints, along with delayed motor development and delayed speech. The molecular basis of the disorder in one patient appears to be in the synthesis of the core region common to xylose-based GAG chains. Decorin, a dermatan sulfate proteoglycan that binds to collagen fibrils, was partially deficient, and some molecules were made without an extended GAG chain. The activity of galactosyl transferase I, the enzyme that adds galactose to xylosylserine, was only 5% of normal in this individual, whereas the parents had 50% of normal activity. The patient′s enzyme was also thermolabile. In addition, galactosyl transferase II, the enzyme responsible for adding the second galactose residue to the GAG chain core, had only 20% of normal activity, and both parents showed reduced activity. Further analysis will be needed to resolve the specific defect, but one possible explanation is that the primary mutation affects the formation or stability of a biosynthetic complex involving several GAG-chain biosynthetic enzymes. The selective effect seen on decorin may reflect substrate preferences.

Three autosomal recessive disorders, diastrophic dystrophy (DTD), atelosteogenesis type II (AOII), and achondrogenesis type IB (ACG-IB), all result from defective cartilage proteoglycan sulfation. These forms of osteochondrodysplasia have various outcomes. AOII and ACG-IB are parinatally lethal due to respiratory insufficiency, whereas DTD patients have symptoms only in the cartilage and bone, including cleft palate, clubbed feet, and other skeletal abnormalities. Those DTD patients surviving infancy often live a nearly normal life span. All of these disorders result from different mutations in the DTD gene that encodes a sulfate transporter. Unlike monosaccharides, sulfate released from degraded macromolecules in the lysosome does not seem to be salvaged well. The heavy demand for sulfate in bone and cartilage proteoglycan synthesis probably explains why the symptoms are most evident in these locations.

Keratan sulfate in the cornea is an N-glycan with polylactosamine repeats (GlcNAcβ1–3Galβ1–4) variably sulfated at the 6-position. Another autosomal recessive disease, macular corneal dystrophy (MCD), causes the cornea to become opaque and corneal lesions develop. Two types of MCD have been described. MCD I appears to be a deficiency in sulfating the repeating units. Both galactose and GlcNAc are sulfated in keratan sulfate, but the enzyme that sulfates galactose in keratan sulfate and GalNAc in chondroitin sulfate is normal in patients. This leaves the GlcNAc 6-sulfotransferase as a more likely candidate for the defect, but this has not yet proven. MCD II differs from MCD I in that the defect in MCD II is not proven, but it may be an allelic form of MCD I.

Future Directions ^(80–83)

It is now clear that a number of syndromes characterized by neonatal presentation of severe neurological and metabolic dysfunction are caused by defects in glycosylation typified by CDGS. It seems likely that an increasing awareness of this constellation of diseases by neonatologists and pediatricians and the availability of a relatively straightforward diagnostic test for these disorders will mean that CDGS will be diagnosed with increasing frequency. To date, only five specific genetic lesions have been identified to account for this disease. However, it seems likely that defects in other genetic loci known to be required for N-glycan synthesis may be found to cause other forms of CDGS. Most of these genes have now been cloned and characterized, and it is likely that the remaining genes will soon be, based in part on successful efforts to isolate these loci in yeast and other organisms, and on the emerging wealth of human DNA sequence information in the public domain EST (expressed sequence tag) databases. Such sequence information will help to define precisely the defect in patients with aberrant transferrin glycoforms suggestive of CDGS and should facilitate prenatal diagnostic efforts. Importantly, as best illustrated by CDGS type Ib and one case of LADII, the ability to discover effective treatments for these devastating disorders will be aided by a clearer understanding of the biochemical pathways involved and through identification of the genetic lesions that account for defects specific to different forms of CDGS. It also seems appropriate to continue to develop models of these diseases via induced mutation approaches in the mouse. These animals provide an opportunity to uncover as yet unknown components of the mammalian glycan synthetic pathways. Moreover, they offer the potential to test therapeutic approaches to the extent that murine deficiencies in glycan synthesis can be shown to accurately reflect the corresponding human pathophysiology.

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