Although the glycosylation defects in the CDGS type 1 serum glycoproteins are well established (section 1.5.4), there have been conflicting reports on the glycosylation defects in the fibroblasts of the CDGS type 1 patients. Comprehensive studies by Powell et al. (1994), Krasnewich et al. (1995) and Panneerselvam and Freeze (1996a) found that the lipid linked oligosaccharides (LLO) produced in CDGS type 1 fibroblasts were truncated. However it had been noted by one group (Krasnewich et al., 1995) that these truncated LLOs were only observed when the cells were labelled for 1 hour with [^H]- mannose, as after a two hour labelling time normal sized LLOs were synthesised. An alternative interpretation of these results is that they indicate that in CDGS type 1 fibroblasts there is a decrease in the rate of synthesis of the LLO. As no evidence of accumulation of truncated LLO was found it is not likely to be an enzyme deficiency in the synthesis the LLO itself, but perhaps in the amount of substrate available in the cell.
The results presented in this chapter in which the LLOs were analysed in the steady state, instead of the rate of synthesis of the LLO, confirmed the hypothesis. The structures of the LLO in the CDGS type 1 fibroblasts in the steady state were similar to that found in normal fibroblasts and no unusual structures were detected. The relative proportions of each structure present were also similar to that found in the normal fibroblasts. Thus
Krasnewich et a i, 1995; Panneerselvam and Freeze, 1996a) were due to the different rate o f synthesis of the LLO in the CDGS type 1 and the normal fibroblasts. The difference between the previous findings and the present investigation can be explained by the difference in the methods that were used.
Ohkura et a l (1997) re-investigated the structures of the LLO in the CDGS type 1 fibroblasts, as they too believed that the truncated LLOs were found as a result of the labelling conditions used and were not indicative of a blockage in the pathway of LLO synthesis. To prove their hypothesis they took a similar approach to the one used in this Chapter. In their case steady state labelling conditions were obtained by incubating the cells with [^H]-mannose for 3 hours instead of the 1 hour used by the other groups (Powell et at., 1994; Krasnewich et al. 1995; Panneerselvam and Freeze, 1996a). In their experiments the LLO in the CDGS type 1 fibroblasts had the same structures as found in the normal fibroblasts, thus confirming the findings reported in this Chapter. These authors also repeated the experiment exchanging [^H]-mannose with [^H]-GlcNAc and again no truncated LLO were found and in both cases, only full size LLO were
transferred to the proteins. In light of these developments it can be concluded that the rate o f synthesis of the LLO is decreased and this is the most probable cause of the under glycosylation of sequons seen in the CDGS type 1 glycoproteins. The decreased rate of synthesis of the LLO is likely to be caused by the deficiency of PMM. In the CDGS type 1 fibroblasts there is a decreased rate of synthesis of mannose-1-phosphate, and so this in turn must lead to decreased rate of synthesis of GDP-mannose and dolichol-P-mannose, both o f which are required for the synthesis o f the LLO (Chapter 1, Figure 1.3).
It was also observed by Ohkura et al. (1997) that the decreased levels of LLO were only observed in the S phase of the cell cycle. The levels of LLO in CDGS type 1 and control fibroblasts were the same in the G1 and G2 phases showing that the difference in levels in CDGS type 1 fibroblasts occurs only in the S phase. The decreased rate of synthesis of LLO only during certain phases of the cell cycle has implications as to the classes of glycoprotein affected. It would be expected that glycoproteins synthesised during the G1 and G2 phases would be normally glycosylated as the amount of LLO available for transfer to the growing polypeptide chain would be sufficient for complete glycosylation.
whereas glycoproteins synthesised during the S phase would suffer from non-occupancy of glycosylation sequons because o f a deficiency of the fully formed oligosaccharide precursor.
It was also found that there was an increase in the amount of dehydrodolichol and a decreased amount of dolichol in CDGS type 1 fibroblasts (Ohkura et a i, 1997). Dolichol is produced from dehydrodolichol by the enzyme dehydrodolichol reductase. An
accumulation of dehydrodolichol in CDGS type 1 (PMM deficient) fibroblasts suggests that there could be a block in the synthesis of dolichol or alternatively it could be the result of a feedback mechanism within the cell due to the lack of utilisation of dolichol as a result of impairment of GDP-mannose production. A deficiency of dehydrodolichol reductase activity has not been shown in CDGS type 1 cell lines as yet.
The methodology o f Powell et al. (1994) and Panneerselvam and Freeze (1996a) only provides a narrow window of time in the glycosylation pathway and does not represent what happens in the steady state. The difference between their results and the present findings can be explained by the fact that with the radiolabelling experiments the rate of production of the oligosaccharide precursor was followed. As there was reduced incorporation o f mannose into the cells a decrease in the rate of production of the oligosaccharide precursor is to be expected. This explanation would account for the production of smaller oligosaccharides after a 1 fiour chase and normal sized
oligosaccharides after a 2 hour chase. Thus in the steady state the presence of the oligosaccharides in the correct proportions is to be expected. The same principle can be applied to the newly synthesised glycoprotein fraction. Using the steady state conditions, the present study has shown that the glycans from CDGS type 1 fibroblasts were the same species and present in the same proportions as was found in normal fibroblasts, whereas Powell et a/. (1994) and Panneerselvam and Freeze (1996a) found smaller glycans in their glycoprotein fraction suggesting that smaller glycans were transferred to the protein.