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1.7.1 Hereditary Fructose Intolerance

Hereditary fructose intolerance (HFI) is another example of a secondary carbohydrate- deficient glycoprotein syndrome. It is caused by a deficiency of fructose-1-phosphate aldolase (aldolase B) (Gitzelman et a i, 1989). Aldolase B cleaves fructose-1-phosphate into D-glyceraldehyde and dihydroxyacetone phosphate in the liver, kidney and small intestine. Aldolases A and C perform similar reactions in the muscle and brain

respectively. These aldolases consist of four subunits and have an overall molecular mass of around 160 kDa. There is a switch from expression of aldolase A and C in fetal liver, kidney and small intestine to aldolase B in later life.

1.7.1.1 Clinical picture, diagnosis and treatment

A child with hereditary fructose intolerance is asymptotic during breast feeding but as soon as weaning occurs with the inclusion of vegetables and fruit in the diet, the symptoms become apparent. They usually consist of poor feeding, vomiting,

hepatomegaly, jaundice and haemorrhage. If left untreated, it can result in death. Babies fed on cow milk formula containing either fructose or sucrose instead of breast feeding show these symptoms immediately. Diagnosis is achieved by measurement of fructose-1- phosphate aldolase B activity in a liver biopsy, as fibroblasts express only aldolase A. Treatment with a fructose- and sucrose-free diet alleviates the symptoms within a few days. Unlike galactosaemic patients, they have no intellectual impairment and lead a normal healthy life (Gitzelman et a i, 1989).

1.7.1.2 Fructose-1-phosphate accumulation and aberrant glycosylation

Many of the toxic effects of fructose in HFI are attributed to depletion o f ATP and inorganic phosphates. However, the build up of fructose-1-phosphate can lead to the inhibition of phosphomannose isomerase and phosphorylase a (Gitzelman et a l, 1989).

It has been recently reported that serum transferrin from patients with HFI is also aberrantly glycosylated like that of CDGS type 1 patients (Adamowicz, 1996). On a normal diet 31.7 % of the serum transferrin from an HFI patient consisted o f asialo-, monosialo-, and disialotransferrin isoforms, compared to 2.3 - 8.6 % for controls and 62.1 % for CDGS type 1. After four months treatment with a fructose-free diet, this value returned to that of the controls. The abnormal transferrin pattern was not found in the parents of this patient. HFI is now considered to be a secondary form o f CDGS (Adamowicz^ 1996).

Jaeken et a/. (1996) have shown that fructose-1 -phosphate is a competitive inhibitor of phosphomannose isomerase (EC 5.3.1.8). Phosphomannose isomerase converts fructose- 6-phosphate to mannose-6-phosphate in the pathway depicted in Figure 1.13. When glucose is the only available carbon source, the PMI reaction is vital in the production o f mannose-6-phosphate, GDP-Man and dolichol-P-Man for the synthesis of

glycoproteins. Patients on a fructose free diet show normal glycosylation o f serum transferrin (Adamowicz et al., 1996). It appears that a depletion in the production of fructose-1-phosphate as a result of removal o f dietary fructose allows normal PMI activity and the production of the mannose-1 -phosphate precursor required for the synthesis of G DP-M an.

1.7.2 Hereditary erythroblastic multinuclearity with a positive acidified

serum lysis test (HEMPAS)

HEMP AS is an autosomal recessive disorder in which the main symptoms include life­ long anaemia, liver cirrhosis, hepatomegaly, splenomegaly, diabetes and gall stones. The major biochemical feature apart from the positive acidified serum lysis test is the

abnormal glycosylation of the erythrocyte band 3 glycoproteins. Band 3 glycans are normally composed of a biantennary trimannosyl core with a polylactosamine side chain.

However, in HEMPAS patients, the band 3 glycans do not contain a polylactosamine side chain on the a 1-6 branch (Fukuda et a l , 1987). The absence of a polylactosamine side chain in the band 3 glycoprotein causes clustering because of increased

hydrophobicity. This abnormal distribution o f band 3 may be the cause of the morphological changes that are seen in the erythrocyte membranes (Fukuda, 1990; Fukuda et a l, 1990). The aberrant glycosylation of band 3 could arise from either a deficiency of a-mannosidase II or N-acetylglucosaminyl transferase II (Fukuda, 1990). Deficiencies o f N-acetylglucosaminyl transferase II (Fukuda et a l, 1987) and a -

mannosidase II (Fukuda et a l, 1990) have been reported, demonstrating that HEMPAS is a heterogeneous genetic disorder. It has also been noted that mice with a deficiency of a-mannosidase II have a clinical phenotype that resembles HEMPAS (Chui et a i, 1997).

The enzyme deficiency in HEMPAS patients has also been found to affect the glycosylation of serum glycoproteins. Analysis o f the glycans released from serum transferrin revealed that many were o f the high mannose and hybrid type rather than disialylated biantennary glycans. a 1-Acid glycoprotein was similarly aberrantly

glycosylated (Fukuda et a l , 1992). Therefore it is likely that the aberrant glycosylation of serum glycoproteins leads to the liver cirrhosis in many patients. Although it would seem that HEMPAS maybe closely related to CDGS type H, HEMPAS differs in that both O-linked and N-linked oligosaccharides are affected (Tomita and Parker, 1994). CDGS type II erythrocytes do not show the typical polylactosamine pattern of HEMPAS erythrocytes and the clinical phenotypes o f the two disorders are not similar (Charuk et a l, 1995).

1.7.3 Rheumatoid arthritis

There is a change in the population of the oligosaccharides attached to IgG in patients with rheumatoid arthritis (RA) (Parekh et a l , 1985; Rademacher et a l, 1988; Axford et a l, 1992). Normal IgG has one conserved glycosylation site at Asn 297 in the Fc region and it is also glycosylated at non-conserved positions in the variable region (Fab). There are no disialylated glycans and only small amounts of monosialylated glycans and glycans bisected by a GlcNAc residue on the glycan in the Fc region. This is in contrast to the glycans present on the Fab region in which there are many disialylated biantennary

glycans, monosialylated and bisected glycans. The oligosaccharides present in IgG in RA

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have a decreased amount of galactose and an increased amount of^N-acetylglucosamine. The same agalactosylated IgG is also observed in the juvenile form of RA and in patients with tuberculosis and Crohn’s disease (Rademacher et al., 1988). The molecular basis of the agalactosylated glycans is thought to be a deficiency of a specific

galactosyltransferase that transfers galactose to agalactosyl IgG (Axford et at., 1987). However, the presence of agalactosyl IgG in Crohn’s disease and tuberculosis suggests that mycobacterium could play a role in the aetiology of RA (Rademacher et a i , 1988). It is interesting to note that serum IgAl from RA patients does not contain

agalactosylated oligosaccharides. Thus it would seem that the agalactosylation defect is limited to IgG. It is not known whether there is an increase in the amount of

agalactosylated IgAl in Crohn’s disease or in tuberculosis (Field et a i , 1994). t

1.7.4 Alcoholism

The carbohydrate-deficient transferrin isoforms that are found in chronic alcoholism are similar to those found in CDGS type 1 in that the disialo and asialo isoforms are formed by the loss of one and two disialylated biantennary glycans respectively (Landberg et a i ,

1995). The proportions of the disialotransferrin and asialotransferrin isoforms are greater in CDGS type 1 than in chronic alcoholism (Stibler et a i , 1991b). However the

underlying mechanisms of non-occupancy of glycosylation sequons in chronic alcoholism remain to be elucidated. Ethanol is oxidised in the liver to produce acef-aldehyde and so it is possible that acet- aldehyde may inhibit the glycosyltransferases, the nucleotide sugar transporters or oligosaccharyltransferase (Marinari et a i , 1993). Although acet-aldehyde has been shown to inhibit galactosyltransferase (Guasch et al., 1992), it must also be a more potent inhibitor of one of the enzymes which acts earlier in the biosynthetic pathway because the glycans that are attached to serum transferrin from alcoholic patients are processed normally (Landberg et al., 1995).

1.8 Aims of the thesis