Clinical and biochemical features in a Congolese infant with congenital disorder of glycosylation (CDG)-IIx.

We describe an infant girl with psychomotor retardation, growth retardation, mild facial dysmorphy, evidence of liver involvement and a type 2 pattern of serum sialotransferrins. Serum transferrin glycan analysis with MALDI-TOF showed an extremely altered N-glycan pattern with a large number of truncated asialoglycans pointing to a severely defective N-glycan processing. The basic defect in this patient with CDG-IIx has not yet been identified.

Congenital disorders of glycosylation: the rapidly growing tip of the iceberg.

In addition to many other organs, the brain is affected in 10 of the 11 known congenital disorders of N-linked glycosylation, mostly to a severe degree. Because a large number of enzymes, transporters and other proteins are involved in glycosylation (both N-linked and O-linked), it is expected that the great majority of congenital disorders of glycosylation (CDG) are yet to be identified. Many neurological patients with a CDG escape diagnosis for that reason, but also because existing screening methods fail to detect all patients with a known CDG. These disorders should be looked for in any patient, regardless of age, with an unexplained neurological disorder.

Oxidative catabolism of alpha-tocopherol in rat liver microsomes.

The goal of this study was to clarify the mechanism responsible for the catabolism of alpha-tocopherol. The vitamin, bound to albumin, was incubated with rat liver microsomes and appeared to be broken down. Optimal production of the metabolite was obtained when 1 mg of microsomal protein was incubated with 36 microM of alpha-tocopherol in the presence of 1.5 mM of NADPH. Chromatographic and mass spectrometric analyses of the metabolite led to the conclusion that it consists of an omega-acid with an opened chroman ring, although we could not perform nuclear magnetic resonance analysis to confirm this. Our data show that alpha-tocopherol is omega-oxidized to a carboxylic acid and that this process can occur in rat liver microsomes in the presence of NADPH and O2. The oxidation to the quinone structure appears to be a subsequent event that may be artifactual and/or catalyzed by a microsomal enzyme(s).

Small intestinal brush border enzymes in cystic fibrosis.

The study concerns the maltase, saccharase, lactase and alkaline phosphatase activity in small intestinal biopsy specimens from 61 consecutively admitted, untreated, Caucasian cystic fibrosis patients. A group of 319 age matched controls admitted during the same time period for undefined gastrointestinal or nutritional disorders acted as the controls. In order to eliminate morphological damage as a confounding factor, the enzyme activities were studied in small intestinal biopsy specimens having both normal stereomicroscopic and histological features. It was shown that neither maltase nor saccharase activity was different in the two groups, in contrast to lactase and alkaline phophatase activity, that was significantly lower in cystic fibrosis patients. The differences could not be explained by the nutritional status as judged by the body mass index. Lactase activity is known to be easily affected by numerous enteropathies. As the information on alkaline phosphatase activity is limited, the low activity is discussed in more detail. Taking into account the literature data, the low alkaline phosphatase activity is tentatively attributed either to enhanced release from the brush border or to the faulty handling of alkaline phophatase protein in the post-golgi compartments secondary to the accumulation of incorrectly glycosylated CFTR in the same cell structures.

Carbohydrate-deficient glycoprotein syndrome type IA (phosphomannomutase-deficiency).

The carbohydrate-deficient glycoprotein or CDG syndromes (OMIM 212065) are a recently delineated group of genetic, multisystem diseases with variable dysmorphic features. The known CDG syndromes are characterized by a partial deficiency of the N-linked glycans of secretory glycoproteins, lysosomal enzymes, and probably also membranous glycoproteins. Due to the deficiency of terminal N-acetylneuraminic acid or sialic acid, the glycan changes can be observed in serum transferrin or other glycoproteins using isoelectrofocusing with immunofixation as the most widely used diagnostic technique. Most patients show a serum sialotransferrin pattern characterized by increased di- and asialotransferrin bands (type I pattern). The majority of patients with type I are phosphomannomutase deficient (type IA), while in a few other patients, deficiencies of phosphomannose isomerase (type IB) or endoplasmic reticulum glucosyltransferase (type IC) have been demonstrated. This review is an update on CDG syndrome type IA.

A novel carbohydrate-deficient glycoprotein syndrome characterized by a deficiency in glucosylation of the dolichol-linked oligosaccharide.

Carbohydrate-deficient glycoprotein syndromes (CDGS) type I are a group of genetic diseases characterized by a deficiency of N-linked protein glycosylation in the endoplasmic reticulum. The majority of these CDGS patients have phosphomannomutase (PMM) deficiency (type A). This enzyme is required for the synthesis of GDP-mannose, one of the substrates in the biosynthesis of the dolichol-linked oligosaccharide Glc3Man9GlcNAc2. This oligosaccharide serves as the donor substrate in the N-linked glycosylation process. We report on the biochemical characterization of a novel CDGS type I in fibroblasts of four related patients with normal PMM activity but a strongly reduced ability to synthesize glucosylated dolichol-linked oligosaccharide leading to accumulation of dolichol-linked Man9GlcNAc2. This deficiency in the synthesis of dolichol-linked Glc3Man9GlcNAc2 oligosaccharide explains the hypoglycosylation of serum proteins in these patients, because nonglucosylated oligosaccharides are suboptimal substrates in the protein glycosylation process, catalyzed by the oligosaccharyltransferase complex. Accordingly, the efficiency of N-linked protein glycosylation was found to be reduced in fibroblasts from these patients.

Lysosomal enzyme activities in serum and leukocytes from patients with carbohydrate-deficient glycoprotein syndrome type IA (phosphomannomutase deficiency).

From 10 patients with carbohydrate-deficient glycoprotein (CDG) syndrome due to phosphomannomutase (PMM) deficiency, out of 10 lysosomal enzymes, 7 enzyme activities were measured in serum and 9 in leukocytes. In serum there was a 2-fold to 4-fold increase in activity of beta-glucuronidase, beta-hexosaminidase, beta-galactosidase, and arylsulphatase A. In leukocytes, however, several enzymes had reduced activity, particularly alpha-fucosidase, beta-glucuronidase and alpha-mannosidase. These abnormalities could result from missorting, defective reuptake and/or reduced stability of the enzymes due to the defective glycosylation.

Determination of glycan structures and molecular masses of the glycovariants of serum transferrin from a patient with carbohydrate deficient syndrome type II.

Serum transferrin from a child with carbohydrate deficient syndrome type II was isolated by immunoaffinity chromatography and separated into minor and major fractions by fast protein liquid chromatography. The structure of the glycans released from the major fraction by hydrazinolysis was established by application of methanolysis and 1H-NMR spectroscopy. The results led to the identification of an N-acetyllactosamininic type monosialylated, monoantennary Man(alpha1-3) linked glycan. By electrospray-mass spectrometry analysis, the whole serum transferrin was separated into at least seven species (I to VII) with molecular masses ranging from 77,958 to 79,130 Da. On the basis of a polypeptide chain molecular mass of 75,143 Da, it was calculated that the major transferrin species III (78,247 Da) contains two monosialylated monoantennary glycans. The molecular mass of transferrin species V and VI (78,678 and 78,971 Da) suggests that one of their two glycans contains an additional N-acetyllactosamine and a sialylated N-acetyllactosamine units, respectively. Transferrin species I and V were found to correspond to the desialylated forms of species III and VI. The abnormal glycan structures can be explained by a defect in the N-acetylglucosaminyltransferase II activity [Charuk et al. (1995) Eur J Biochem 230: 797-805].

3-Phosphoglycerate dehydrogenase deficiency: an inborn error of serine biosynthesis.

Serine concentrations were markedly decreased in the cerebrospinal fluid of two brothers with congenital microcephaly, profound psychomotor retardation, hypertonia, epilepsy, growth retardation, and hypogonadism. The youngest boy also had congenital bilateral cataract. Magnetic resonance imaging of the brain showed evidence of dysmyelination. Plasma serine as well as plasma and cerebrospinal fluid glycine concentrations were also decreased but to a lesser extent. Treatment with oral serine in the youngest patient significantly increased cerebrospinal fluid serine and abolished the convulsions. In fibroblasts of both patients, a decreased activity was demonstrated of 3-phosphoglycerate dehydrogenase, the first step of serine biosynthesis (22% and 13% of the mean control value). This is an unusual disorder as the great majority of aminoacidopathies are catabolic defects. It is a severe but potentially treatable inborn error of metabolism that has not been previously reported in man.

Peroxisomal beta-oxidation. Purification of four novel 3-hydroxyacyl-CoA dehydrogenases from rat liver peroxisomes.

Peroxisomes are capable of beta-oxidizing a variety of substrates including the CoA esters of straight chain fatty acids, 2-methyl-branched fatty acids and the bile acid intermediates di- and trihydroxycoprostanic acids. The first reaction of peroxisomal beta-oxidation is catalyzed by an acyl-CoA oxidase. Rat liver peroxisomes contain three acyl-CoA oxidases: 1) palmitoyl-CoA oxidase, oxidizing straight chain acyl-CoAs; 2) pristanoyl-CoA oxidase, oxidizing 2-methyl-branched acyl-CoAs; and 3) trihydroxycoprostanoyl-CoA oxidase, oxidizing the CoA esters of the bile acid intermediates (Van Veldhoven, P.P., Vanhove, G., Asselberghs, S., Eyssen, H. J., and Mannaerts, G. P. (1992) J. Biol. Chem. 267, 20065-20074). We have now investigated whether the third step of peroxisomal beta-oxidation, catalyzed by a 3-hydroxyacyl-CoA dehydrogenase, is also catalyzed by multiple enzymes, using the 3-hydroxyacyl-CoA derivatives of palmitic acid, 2-methylpalmitic acid, and trihydroxycoprostanic acid as the substrates to monitor the dehydrogenase activities. In order to avoid contamination with mitochondrial 3-hydroxyacyl-CoA dehydrogenases, highly purified peroxisomes from untreated rats were employed as the enzyme source. Subfractionation of the peroxisomes revealed that the major portion of the dehydrogenase activities with all three substrates was present in the peripheral membrane protein fraction. Separation of this fraction on various chromatographic columns resulted in the purification of the well known multifunctional protein, a 78-kDa monomeric protein that displays 3-hydroxyacyl-CoA dehydrogenase plus hydratase activity, as well as of four additional novel dehydrogenases with different substrate specificities. Three of the enzymes are monomeric proteins of 35 kDa, 56 kDa, and 79 kDa, respectively. The latter enzyme also displays hydratase activity. The fourth enzyme is a dimer of 89 kDa, the subunits of which form a doublet at 40 kDa. The exact physiological role of each of the 3-hydroxyacyl-CoA dehydrogenases requires further investigation.

Carbohydrate deficient glycoprotein syndrome type II: a deficiency in Golgi localised N-acetyl-glucosaminyltransferase II.

The carbohydrate deficient glycoprotein (CDG) syndromes are a family of genetic multisystemic disorders with severe nervous system involvement. This report is on a child with a CDG syndrome that differs from the classical picture but is very similar to a patient reported in 1991. Both these patients are therefore designated CDG syndrome type II. Compared with type I patients they have a more severe psychomotor retardation but no peripheral neuropathy nor cerebellar hypoplasia. The serum transferrin isoform pattern obtained by isoelectric focusing showed disialotransferrin as the major fraction. The serum disialotransferrin, studied in the present patient, contained two moles of truncated monoantennary Sialyl-Gal-GlcNAc-Man(alpha 1-->3)[Man(alpha 1-->6)]Man(beta 1-->4)GlcNAc (beta 1-->4)GlcNAc-Asn per mole of transferrin. A profoundly deficient activity of the Golgi enzyme N-acetylglucosaminyltransferase II (EC 2.4.1.143) was demonstrated in fibroblasts.

The carbohydrate-deficient glycoprotein syndromes: pre-Golgi and Golgi disorders?

The carbohydrate-deficient glycoprotein syndromes are a recently delineated group of genetic, multisystemic diseases with major nervous system involvement. Three distinct variants have been recognized and there are probably many more. They are characterized by a deficiency of the carbohydrate moiety of secretory glycoproteins, lysosomal enzymes and probably also membranous glycoproteins. The biochemical changes are most readily observed in serum transferrin and the diagnosis is usually made by isoelectric focusing of this glycoprotein. The deficiency of sialic acid, in particular, results in a cathodal shift and hence the presence of abnormal isoforms of transferrin with higher isoelectric points than normal. The basic defects are probably in the processing and synthesis of the carbohydrate moiety of glycoproteins; there is indirect evidence for a deficiency of asparagine-N-linked oligosaccharide transfer in type I (endoplasmic reticulum defect) and for a deficiency of N-acetylglucosaminyltransferase II in type II (Golgi defect). From the large number of patients detected in only a few years, it is expected that these diseases will become as important as, for example, the lysosomal, peroxisomal or mitochondrial disorders. Their study will undoubtedly yield a wealth of new information on the function of glycoproteins and of their carbohydrate moiety.